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44 56 binding-site site Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger TITLE
74 98 sodium-calcium exchanger protein_type Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger TITLE
0 19 Na+/Ca2+ exchangers protein_type Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT
32 35 Na+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT
113 117 Ca2+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT
146 150 Ca2+ chemical Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. ABSTRACT
92 111 structural analysis experimental_method Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
119 128 exchanger protein_type Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
134 158 Methanococcus jannaschii species Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
160 166 NCX_Mj protein Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
168 176 bound to protein_state Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
177 180 Na+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
182 186 Ca2+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
190 194 Sr2+ chemical Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
228 231 apo protein_state Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. ABSTRACT
130 133 Na+ chemical This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. ABSTRACT
135 139 Ca2+ chemical This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism. ABSTRACT
44 80 conformational free-energy landscape evidence An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT
84 90 NCX_Mj protein An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT
104 117 ion-occupancy protein_state An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT
135 183 enhanced-sampling molecular-dynamics simulations experimental_method An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT
207 225 crystal structures evidence An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. ABSTRACT
6 18 calculations experimental_method These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT
58 65 outward protein_state These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT
69 75 inward protein_state These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT
182 185 Na+ chemical These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT
186 190 Ca2+ chemical These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport. ABSTRACT
0 19 Na+/Ca2+ exchangers protein_type Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO
21 24 NCX protein_type Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO
66 70 Ca2+ chemical Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. INTRO
0 3 NCX protein_type NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO
52 56 Ca2+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO
138 141 Na+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO
166 169 Na+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO
175 179 Ca2+ chemical NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry. INTRO
17 20 NCX protein_type The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport. INTRO
47 50 NCX protein_type The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO
67 93 membrane-spanning segments structure_element The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO
121 127 halves structure_element The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. INTRO
14 20 halves structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
32 48 highly conserved protein_state Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
72 80 α-repeat structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
142 152 eukaryotic taxonomy_domain Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
153 156 NCX protein_type Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
166 172 halves structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
198 229 intracellular regulatory domain structure_element Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
240 246 absent protein_state Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
250 259 microbial taxonomy_domain Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
260 263 NCX protein_type Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1). INTRO
91 94 NCX protein_type Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information. INTRO
29 38 structure evidence Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO
42 48 NCX_Mj protein Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO
54 78 Methanococcus jannaschii species Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO
138 141 NCX protein_type Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. INTRO
5 14 structure evidence This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
25 34 exchanger protein_type This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
41 55 outward-facing protein_state This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
95 112 ion-binding sites site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
126 134 internal site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
136 140 Sint site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
143 151 external site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
153 157 Sext site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
160 172 Ca2+-binding site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
174 177 SCa site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
183 189 middle site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
191 195 Smid site This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
241 254 occluded from protein_state This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b). INTRO
53 63 eukaryotic taxonomy_domain With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO
78 84 NCX_Mj protein With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO
247 250 NCX protein_type With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX. INTRO
43 53 structures evidence In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
57 71 outward-facing protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
72 81 wild-type protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
82 88 NCX_Mj protein In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
89 104 in complex with protein_state In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
105 108 Na+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
110 114 Ca2+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
119 123 Sr2+ chemical In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations. INTRO
6 16 structures evidence These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment. INTRO
33 63 molecular-dynamics simulations experimental_method An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. INTRO
86 96 structures evidence An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. INTRO
6 18 calculations experimental_method These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO
66 80 outward-facing protein_state These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO
81 90 exchanger protein_type These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO
143 156 inward-facing protein_state These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism. INTRO
14 17 Na+ chemical Extracellular Na+ binding RESULTS
27 48 central binding sites site The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
87 93 NCX_Mj protein The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
94 103 structure evidence The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
141 144 Na+ chemical The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
149 153 Ca2+ chemical The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
169 177 crystals evidence The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals. RESULTS
73 76 Na+ chemical To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. RESULTS
110 114 Ca2+ chemical To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. RESULTS
0 8 Crystals evidence Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS
30 34 NaCl chemical Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS
45 64 lipidic cubic phase experimental_method Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS
66 69 LCP experimental_method Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. RESULTS
41 44 LCP experimental_method The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS
132 136 NaCl chemical The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS
141 145 EGTA chemical The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). RESULTS
0 17 X-ray diffraction experimental_method X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
34 42 crystals evidence X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
54 57 Na+ chemical X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
85 114 electron-density distribution evidence X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
124 128 Sext site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
130 133 SCa site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
138 142 Sint site X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
157 160 Na+ chemical X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c). RESULTS
0 20 Occupancy refinement experimental_method Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
35 38 Na+ chemical Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
52 56 Sint site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
61 64 SCa site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
72 75 Na+ chemical Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
131 135 Sint site Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1). RESULTS
19 22 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
26 30 Sext site Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
70 77 density evidence Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
106 109 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
130 134 Sext site Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
174 177 Na+ chemical Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c). RESULTS
4 7 Na+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS
22 25 SCa site The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS
57 60 Na+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS
62 66 Ca2+ chemical The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS
121 125 Smid site The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. RESULTS
41 46 water chemical Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS
70 73 Na+ chemical Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS
87 91 Smid site Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS
122 132 simulation experimental_method Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. RESULTS
11 27 electron density evidence First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS
31 35 Smid site First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS
73 76 Na+ chemical First, the electron density at Smid does not depend significantly on the Na+ concentration. RESULTS
45 49 Smid site Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). RESULTS
76 79 Na+ chemical Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). RESULTS
4 9 water chemical The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
22 26 Smid site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
33 47 hydrogen-bonds bond_interaction The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
57 73 highly conserved protein_state The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
74 79 Glu54 residue_name_number The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
84 90 Glu213 residue_name_number The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
158 168 coordinate bond_interaction The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
178 181 Na+ chemical The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
190 194 Sext site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
196 199 SCa site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
204 208 Sint site The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint. RESULTS
45 50 Glu54 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
55 61 Glu213 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
81 87 Asp240 residue_name_number It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
102 106 Smid site It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
127 130 Asn residue_name It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
134 144 eukaryotic taxonomy_domain It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
145 148 NCX protein_type It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
159 169 protonated protein_state It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
206 216 simulation experimental_method It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study. RESULTS
0 3 Na+ chemical Na+-dependent conformational change RESULTS
4 10 NCX_Mj protein The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS
11 21 structures evidence The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS
33 36 Na+ chemical The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS
69 72 Na+ chemical The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS
84 88 Sext site The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2). RESULTS
5 8 Na+ chemical When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
18 22 Sext site When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
26 30 high protein_state When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
51 66 N-terminal half structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
70 73 TM7 structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
91 104 short helices structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
106 110 TM7a structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
115 119 TM7b structure_element When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a). RESULTS
0 4 TM7b structure_element TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS
23 44 central binding sites site TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS
103 109 Ala206 residue_name_number TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS
110 122 coordinating bond_interaction TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS
127 130 Na+ chemical TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). RESULTS
14 18 Sext site However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
27 32 empty protein_state However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
36 39 low protein_state However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
40 43 Na+ chemical However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
60 64 TM7a structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
69 73 TM7b structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
103 108 helix structure_element However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
146 152 Ala206 residue_name_number However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d). RESULTS
0 4 TM7a structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS
16 36 hydrophobic contacts bond_interaction TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS
46 61 C-terminal half structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS
65 68 TM6 structure_element TM7a also forms hydrophobic contacts with the C-terminal half of TM6. RESULTS
33 42 structure evidence These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS
48 51 Na+ chemical These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS
55 59 Sext site These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS
107 114 helices structure_element These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). RESULTS
38 41 TM6 structure_element This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS
46 49 TM1 structure_element This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS
138 149 transporter protein_type This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS
166 179 inward-facing protein_state This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation. RESULTS
21 26 TM7ab structure_element The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
84 88 Sext site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
93 97 Smid site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
105 108 SCa site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
113 117 Sint site The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
125 133 occluded protein_state The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d). RESULTS
10 20 structures evidence Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
24 28 high protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
33 36 low protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
37 40 Na+ chemical Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
70 84 outward-facing protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
85 93 occluded protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
98 112 partially open protein_state Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. RESULTS
47 50 Na+ chemical This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS
64 68 Sext site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS
85 88 Na+ chemical This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS
106 110 Sint site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS
115 118 SCa site This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. RESULTS
4 41 crystallographic titration experiment experimental_method Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
61 65 K1/2 evidence Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
74 77 Na+ chemical Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
145 149 Sext site Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
153 171 partially occupied protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
180 186 NCX_Mj protein Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
187 194 crystal evidence Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
220 228 occluded protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
233 247 partially open protein_state Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations. RESULTS
26 38 Na+ affinity evidence This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS
69 72 Na+ chemical This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS
100 103 NCX protein_type This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS
118 128 eukaryotes taxonomy_domain This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. RESULTS
21 24 Na+ chemical The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
105 116 transporter protein_type The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
150 166 Hill coefficient evidence The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
174 177 Na+ chemical The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
210 220 eukaryotic taxonomy_domain The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
221 224 NCX protein_type The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX. RESULTS
14 18 Ca2+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS
23 27 Sr2+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS
63 66 Na+ chemical Extracellular Ca2+ and Sr2+ binding and their competition with Na+ RESULTS
17 21 Ca2+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS
31 37 NCX_Mj protein To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS
56 59 Na+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS
70 91 titrated the crystals experimental_method To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS
97 101 Sr2+ chemical To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). RESULTS
0 4 Sr2+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS
23 26 NCX protein_type Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS
40 44 Ca2+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS
75 78 Na+ chemical Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS
94 120 electron-density intensity evidence Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. RESULTS
0 23 Protein crystals soaked experimental_method Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
35 39 Sr2+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
51 54 Na+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
73 94 electron-density peak evidence Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
103 106 SCa site Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
139 143 Sr2+ chemical Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a). RESULTS
4 15 Sr2+-loaded protein_state The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS
16 22 NCX_Mj protein The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS
23 32 structure evidence The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS
44 58 partially open protein_state The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS
88 91 Na+ chemical The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. RESULTS
11 15 Sr2+ chemical Binding of Sr2+, however, excludes Na+ entirely. RESULTS
35 38 Na+ chemical Binding of Sr2+, however, excludes Na+ entirely. RESULTS
0 18 Crystal titrations experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
24 34 decreasing experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
35 39 Sr2+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
43 53 increasing experimental_method Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
54 57 Na+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
76 80 Sr2+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
94 108 outward-facing protein_state Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
109 115 NCX_Mj protein Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
170 173 Na+ chemical Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b). RESULTS
16 19 Na+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
30 34 Sr2+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
36 39 Na+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
60 64 Sr2+ chemical Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
88 94 NCX_Mj protein Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
102 112 Na+-loaded protein_state Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
114 128 fully occluded protein_state Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state. RESULTS
8 29 titration experiments experimental_method Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
42 46 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
51 55 Sr2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
67 73 NCX_Mj protein Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
100 129 electron density distribution evidence Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
135 153 crystals soaked in experimental_method Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
154 158 high protein_state Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
159 163 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
168 171 low protein_state Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
172 175 Na+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
192 196 Ca2+ chemical Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
209 213 Smid site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
225 228 SCa site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
252 255 SCa site Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b). RESULTS
11 15 Ca2+ chemical Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS
113 118 water chemical Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS
145 157 coordinating bond_interaction Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b). RESULTS
4 11 partial protein_state The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
12 16 Ca2+ chemical The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
17 26 occupancy protein_state The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
30 34 Smid site The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
55 61 Asp240 residue_name_number The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
107 117 coordinate bond_interaction The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
118 122 Ca2+ chemical The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. RESULTS
9 45 functional and computational studies experimental_method Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS
65 71 Asp240 residue_name_number Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS
80 90 protonated protein_state Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. RESULTS
16 19 NCX protein_type Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
29 35 Asp240 residue_name_number Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
39 50 substituted experimental_method Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
54 57 Asn residue_name Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
97 101 Ca2+ chemical Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
113 117 Smid site Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. RESULTS
0 3 SCa site SCa is therefore the functional Ca2+ site. RESULTS
32 41 Ca2+ site site SCa is therefore the functional Ca2+ site. RESULTS
13 17 Sr2+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
19 23 Ca2+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
39 47 affinity evidence Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
51 65 outward-facing protein_state Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
66 72 NCX_Mj protein Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
105 108 Na+ chemical Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c). RESULTS
32 66 physiological and biochemical data evidence This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS
76 86 eukaryotic taxonomy_domain This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS
87 90 NCX protein_type This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS
95 101 NCX_Mj protein This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS
131 144 Ca2+ affinity evidence This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side. RESULTS
18 50 crystallographic titration assay experimental_method Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
61 65 Ca2+ chemical Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
92 100 affinity evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
147 162 Ca2+ affinities evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
196 199 NCX protein_type Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
201 203 Km evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
219 225 NCX_Mj protein Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
227 229 Km evidence Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM). RESULTS
22 51 crystal titration experiments experimental_method Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
78 91 binding sites site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
95 109 outward-facing protein_state Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
110 116 NCX_Mj protein Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
148 152 Sint site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
157 161 Sext site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
166 169 Na+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
187 190 SCa site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
222 226 Ca2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
251 254 Na+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
279 289 simulation experimental_method Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
308 312 Sr2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
314 318 Smid site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
352 356 Ca2+ chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
378 382 Smid site Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
410 415 water chemical Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water. RESULTS
4 21 ion-binding sites site The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
25 31 NCX_Mj protein The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
70 73 Na+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
122 125 Na+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
130 134 Ca2+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
139 143 Sr2+ chemical The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
188 198 eukaryotic taxonomy_domain The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
199 209 exchangers protein_type The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers. RESULTS
2 11 structure evidence A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
15 21 NCX_Mj protein A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
22 29 without protein_state A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
30 33 Na+ chemical A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
37 41 Ca2+ chemical A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
42 47 bound protein_state A structure of NCX_Mj without Na+ or Ca2+ bound RESULTS
3 6 apo protein_state An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS
16 30 outward-facing protein_state An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS
31 37 NCX_Mj protein An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS
140 143 Na+ chemical An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS
158 162 Ca2+ chemical An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM). RESULTS
29 32 apo protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
39 48 structure evidence We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
52 58 NCX_Mj protein We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
63 76 crystallizing experimental_method We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
92 100 lower pH protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
112 122 absence of protein_state We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
123 126 Na+ chemical We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods). RESULTS
5 14 structure evidence This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
33 47 partially open protein_state This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
48 57 structure evidence This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
67 70 Na+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
85 89 Ca2+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
97 101 Sr2+ chemical This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. RESULTS
7 12 TM7ab structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS
28 46 extracellular half structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS
54 57 TM6 structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS
62 65 TM1 structure_element First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS
168 181 binding sites site First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites. RESULTS
8 13 Glu54 residue_name_number Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
18 24 Glu213 residue_name_number Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
58 71 binding sites site Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
91 105 hydrogen-bonds bond_interaction Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
132 148 ion coordination bond_interaction Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
156 172 fully Na+-loaded protein_state Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
173 182 structure evidence Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d). RESULTS
13 26 binding sites site Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS
36 52 fully accessible protein_state Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS
101 117 electron density evidence Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules. RESULTS
5 8 apo protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
9 18 structure evidence This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
49 57 unloaded protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
59 63 open protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
73 87 outward-facing protein_state This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
88 94 NCX_Mj protein This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. RESULTS
20 29 structure evidence Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS
46 62 fully protonated protein_state Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS
76 87 transporter protein_type Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS
98 101 Na+ chemical Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS
106 110 Ca2+ chemical Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. RESULTS
49 69 computer simulations experimental_method Such interpretation would be consistent with the computer simulations reported below. RESULTS
8 24 transport assays experimental_method Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
28 34 NCX_Mj protein Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
63 74 presence of protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
75 78 Na+ chemical Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
82 86 Ca2+ chemical Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
88 94 low pH protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
95 106 inactivates protein_state Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. RESULTS
54 60 NCX_Mj protein Ion occupancy determines the free-energy landscape of NCX_Mj RESULTS
5 34 secondary-active transporters protein_type That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS
214 222 outward- protein_state That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS
227 238 inward-open protein_state That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states. RESULTS
0 3 NCX protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
33 36 Na+ chemical NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
42 46 Ca2+ chemical NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
78 88 antiporter protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
90 100 symporters protein_type NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
217 222 bound protein_state NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
234 237 apo protein_state NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state. RESULTS
64 100 conformational free-energy landscape evidence To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS
104 110 NCX_Mj protein To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS
175 193 molecular dynamics experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS
195 197 MD experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS
199 210 simulations experimental_method To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations. RESULTS
62 71 structure evidence This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS
75 81 NCX_Mj protein This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS
104 128 crystallographic studies experimental_method This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. RESULTS
44 54 structures evidence As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state. RESULTS
136 142 NCX_Mj protein As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state. RESULTS
24 38 MD simulations experimental_method A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS
97 103 NCX_Mj protein A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS
104 113 structure evidence A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS
134 151 ion-binding sites site A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy. RESULTS
23 32 simulated experimental_method Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS
37 53 outward-occluded protein_state Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS
131 139 high-Na+ protein_state Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS
140 157 crystal structure evidence Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b). RESULTS
9 12 Na+ chemical That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
25 29 Sext site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
31 34 SCa site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
40 44 Sint site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
52 56 D240 residue_name_number That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
60 70 protonated protein_state That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
77 82 water chemical That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
101 105 Smid site That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. RESULTS
4 7 Na+ chemical The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time. RESULTS
15 19 Sext site The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time. RESULTS
4 7 Na+ chemical The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS
16 19 SCa site The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS
24 28 Sint site The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS
75 85 simulation experimental_method The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. RESULTS
14 25 simulations experimental_method These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS
61 72 transporter protein_type These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS
116 134 crystal structures evidence These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. RESULTS
45 48 Na+ chemical The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS
54 58 Sext site The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS
84 89 TM7ab structure_element The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). RESULTS
7 10 Na+ chemical When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
20 25 bound protein_state When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
27 32 TM7ab structure_element When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
80 99 α-helical fragments structure_element When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
150 164 hydrogen-bonds bond_interaction When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
173 177 F202 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
182 186 A206 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
192 196 T203 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
201 205 F207 residue_name_number When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b). RESULTS
25 29 Sext site This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS
97 100 Na+ chemical This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS
147 151 A206 residue_name_number This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g). RESULTS
5 9 Sext site With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
10 15 empty protein_state With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
26 31 TM7ab structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
50 57 α-helix structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
111 114 TM3 structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
119 122 TM7 structure_element With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
145 150 water chemical With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
202 206 Sext site With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
233 244 transporter protein_type With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
248 266 no longer occluded protein_state With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded. RESULTS
16 19 Na+ chemical Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS
25 28 SCa site Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS
33 37 Sint site Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). RESULTS
55 58 TM7 structure_element The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS
63 66 TM2 structure_element The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS
151 154 Na+ chemical The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS
162 165 SCa site The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e). RESULTS
0 3 TM1 structure_element TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS
8 11 TM6 structure_element TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS
76 92 outward-occluded protein_state TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). RESULTS
38 53 aqueous channel site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS
76 79 SCa site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS
84 88 Sint site Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). RESULTS
4 15 transporter protein_type The transporter thus becomes fully outward-open. RESULTS
29 47 fully outward-open protein_state The transporter thus becomes fully outward-open. RESULTS
111 120 exchanger protein_type To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS
167 182 MD calculations experimental_method To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS
242 258 outward-occluded protein_state To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS
263 281 fully outward-open protein_state To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS
312 333 free-energy landscape evidence To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods). RESULTS
68 71 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
75 79 Sext site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
81 84 SCa site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
89 93 Sint site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
100 103 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
112 115 SCa site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
120 124 Sint site As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
130 137 without protein_state As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
138 141 Na+ chemical As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+. RESULTS
6 18 calculations experimental_method These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS
40 43 Na+ chemical These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS
67 78 transporter protein_type These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS
108 144 conformational free-energy landscape evidence These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. RESULTS
9 18 Na+ sites site When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
44 63 free-energy minimum evidence When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
168 173 TM7ab structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
205 208 TM2 structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
213 216 TM3 structure_element When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
229 242 binding sites site When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b). RESULTS
40 51 transporter protein_type At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
64 74 metastable protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
76 85 half-open protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
109 114 TM7ab structure_element At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
142 146 Sext site At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
150 154 open protein_state At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b). RESULTS
4 7 Na+ chemical The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
15 19 Sext site The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
28 45 fully coordinated protein_state The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
62 67 water chemical The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
111 115 A206 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
147 151 F202 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
154 158 A206 residue_name_number The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
161 174 hydrogen-bond bond_interaction The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:OA206:N hydrogen-bond (Fig. 5c). RESULTS
5 14 semi-open protein_state This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
91 94 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
109 112 SCa site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
117 121 Sint site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
127 130 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
185 188 Na+ chemical This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
192 196 Sext site This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
212 222 metastable protein_state This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation. RESULTS
92 107 aqueous channel site Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
141 144 SCa site Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
153 156 TM7 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
161 164 TM2 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
178 192 gating helices structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
193 196 TM1 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
201 204 TM6 structure_element Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
219 223 open protein_state Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c). RESULTS
23 44 free-energy landscape evidence Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS
54 72 partially occupied protein_state Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS
101 109 occluded protein_state Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). RESULTS
34 37 Na+ chemical Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
48 51 SCa site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
56 60 Sint site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
82 103 free-energy landscape evidence Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
111 122 transporter protein_type Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
170 180 fully open protein_state Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
205 221 aqueous channels site Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c). RESULTS
22 30 occluded protein_state The transition to the occluded state in this apo state is again energetically unfeasible. RESULTS
45 48 apo protein_state The transition to the occluded state in this apo state is again energetically unfeasible. RESULTS
67 71 open protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
76 85 semi-open protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
132 135 Na+ chemical From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
175 178 Na+ chemical From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
287 308 free-energy landscape evidence From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
316 327 transporter protein_type From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
346 354 occluded protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
392 417 Na+ occupancy is complete protein_state From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete. RESULTS
41 44 Na+ chemical This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
93 103 Ca2+-bound protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
144 155 transporter protein_type This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
223 235 outward-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
276 280 Ca2+ chemical This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
295 303 occluded protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
331 340 semi-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
385 396 inward-open protein_state This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state. RESULTS
26 28 H+ chemical By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS
97 106 semi-open protein_state By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS
133 143 fully open protein_state By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion. RESULTS
42 71 enhanced-sampling simulations experimental_method To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
80 84 Ca2+ protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
89 97 H+-bound protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
108 122 outward-facing protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
123 129 NCX_Mj protein To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
169 172 Na+ chemical To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
249 259 structures evidence To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
267 277 Ca2+-bound protein_state To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted). RESULTS
4 14 calculated experimental_method The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
15 36 free-energy landscape evidence The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
41 51 Ca2+-bound protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
52 58 NCX_Mj protein The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
103 107 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
149 155 NCX_Mj protein The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
176 180 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
186 194 occluded protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
196 206 dehydrated protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
274 283 exchanger protein_type The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
303 312 semi-open protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
317 321 open protein_state The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
356 360 Ca2+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
373 376 Na+ chemical The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
402 425 aqueous access channels site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
439 443 Sext site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
448 451 SCa site The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c). RESULTS
13 24 protonation protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
28 33 Glu54 residue_name_number By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
38 44 Glu213 residue_name_number By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
55 63 occluded protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
133 139 NCX_Mj protein By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
159 166 protons chemical By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
176 184 H+-bound protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
204 213 exchanger protein_type By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
228 237 semi-open protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
267 273 low pH protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
275 278 apo protein_state By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
279 296 crystal structure evidence By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
302 304 H+ chemical By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c). RESULTS
21 54 systematic computational analysis experimental_method Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
58 72 outward-facing protein_state Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
73 79 NCX_Mj protein Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
168 186 Na+/Ca2+ exchanger protein_type Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
253 274 free-energy landscape evidence Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
335 343 occluded protein_state Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible. RESULTS
5 13 occluded protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
74 81 outward protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
86 97 inward-open protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
137 148 dehydration protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
189 207 complete occupancy protein_state This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
215 228 binding sites site This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites. RESULTS
78 85 outward protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS
91 102 inward-open protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS
128 144 secondary-active protein_state The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS
145 156 transporter protein_type The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized. DISCUSS
32 43 antiporters protein_type It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles. DISCUSS
150 169 membrane symporters protein_type It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles. DISCUSS
35 45 antiporter protein_type Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number. DISCUSS
67 77 antiporter protein_type Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS
83 92 symporter protein_type Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS
143 168 alternating-access switch site Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch. DISCUSS
103 121 structural studies experimental_method Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS
126 160 quantitative molecular simulations experimental_method Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS
166 184 Na+/Ca2+ exchanger protein_type Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger. DISCUSS
29 35 NCX_Mj protein Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7). DISCUSS
25 39 outward-facing protein_state The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
40 46 NCX_Mj protein The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
55 68 inward-facing protein_state The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
69 87 crystal structures evidence The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
99 117 Ca2+/H+ exchangers protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
168 171 NCX protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
209 212 TM1 structure_element The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
217 220 TM6 structure_element The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
249 260 transporter protein_type The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter. DISCUSS
56 76 extracellular region structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS
84 91 TM2-TM3 structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS
96 110 TM7-TM8 bundle structure_element Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release. DISCUSS
48 63 N-terminal half structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
67 70 TM7 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
72 77 TM7ab structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
123 126 TM2 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
131 134 TM3 structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
151 156 TM7ab structure_element The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
213 229 aqueous channels site The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
247 264 ion-binding sites site The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution. DISCUSS
30 33 TM7 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
71 88 ion-binding sites site Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
123 126 TM6 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
145 148 TM6 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
153 156 TM1 structure_element Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
180 193 inward-facing protein_state Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation. DISCUSS
4 22 crystal structures evidence The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
26 32 NCX_Mj protein The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
60 64 Na+, chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
65 70 Ca2+, chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
71 75 Sr2+ chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
79 81 H+ chemical The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
82 87 bound protein_state The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
101 110 exchanger protein_type The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. DISCUSS
115 126 transporter protein_type These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions. DISCUSS
20 27 crystal evidence For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
31 37 NCX_Mj protein For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
41 44 LCP experimental_method For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
50 68 extracellular half structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
76 90 gating helices structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
92 95 TM6 structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
100 103 TM1 structure_element For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
190 207 ion-binding sites site For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
235 238 apo protein_state For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
240 246 low pH protein_state For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
247 256 structure evidence For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure). DISCUSS
17 50 calculated free-energy landscapes evidence Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states. DISCUSS
134 157 crystallized structures evidence Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states. DISCUSS
4 15 simulations experimental_method The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event. DISCUSS
58 66 occluded protein_state Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS
130 136 NCX_Mj protein Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS
146 149 Na+ chemical Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS
151 155 Ca2+ chemical Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry. DISCUSS
89 101 transporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS
140 150 symporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS
155 166 antiporters protein_type We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS
197 205 occluded protein_state We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS
228 272 protein conformational free-energy landscape evidence We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms. DISCUSS
122 128 NCX_Mj protein In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. DISCUSS
137 147 eukaryotic taxonomy_domain In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. DISCUSS
48 70 ion-binding affinities evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS
89 116 crystallographic titrations experimental_method The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS
125 127 Km evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS
132 143 K1/2 values evidence The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS
168 185 functional assays experimental_method The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above. DISCUSS
113 119 NCX_Mj protein Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS
132 135 NCX protein_type Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS
193 199 NCX_Mj protein Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS
200 210 structures evidence Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d). DISCUSS
4 25 crystallographic data evidence The crystallographic data also provides the long-sought structural basis for thetwo-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS
149 159 eukaryotic taxonomy_domain The crystallographic data also provides the long-sought structural basis for thetwo-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS
160 163 NCX protein_type The crystallographic data also provides the long-sought structural basis for thetwo-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS
212 218 NCX_Mj protein The crystallographic data also provides the long-sought structural basis for thetwo-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS
237 255 Na+/Ca2+ exchanger protein_type The crystallographic data also provides the long-sought structural basis for thetwo-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger. DISCUSS
18 36 crystal titrations experimental_method Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
66 69 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
70 74 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
91 95 Sint site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
100 103 SCa site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
111 115 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
120 123 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
162 165 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
211 214 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
239 242 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
261 266 water chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
280 284 Smid site Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
313 317 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
334 350 Hill coefficient evidence Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
361 364 Na+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
389 393 Ca2+ chemical Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes. DISCUSS
4 8 Sext site The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
51 66 activation site site The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
78 81 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
127 130 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
149 152 Na+ chemical The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
199 207 occluded protein_state The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state. DISCUSS
26 30 Ca2+ chemical Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. DISCUSS
34 38 Smid site Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. DISCUSS
8 18 structures evidence Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
22 28 NCX_Mj protein Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
29 37 bound to protein_state Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
38 42 Cd2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
46 50 Mn2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
104 108 Smid site Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
123 127 Sr2+ chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
142 145 SCa site Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
182 185 NCX protein_type Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
199 206 calcium chemical Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium. DISCUSS
37 43 NCX_Mj protein Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
72 76 Ca2+ chemical Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
80 83 Na+ chemical Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
147 174 hydrogen-deuterium exchange experimental_method Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
176 179 HDX experimental_method Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
184 190 NCX_Mj protein Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
199 207 presence protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
211 221 absence of protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
259 273 outward-facing protein_state Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations. DISCUSS
36 40 Ca2+ chemical Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS
44 47 Na+ chemical Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS
89 97 HDX rate evidence Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS
132 148 α-repeat helices structure_element Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. DISCUSS
231 248 ion-binding sites site We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state. DISCUSS
80 88 occluded protein_state In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate. DISCUSS
194 202 HDX rate evidence In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate. DISCUSS
70 78 HDX rate evidence Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS
97 100 Na+ chemical Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS
105 109 Ca2+ chemical Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations. DISCUSS
7 40 calculated free-energy landscapes evidence As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
47 50 Na+ chemical As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
55 59 Ca2+ chemical As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
88 99 transporter protein_type As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
136 145 ion-bound protein_state As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
219 229 fully open protein_state As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates. DISCUSS
0 3 Na+ chemical Na+ binding to outward-facing NCX_Mj. FIG
15 29 outward-facing protein_state Na+ binding to outward-facing NCX_Mj. FIG
30 36 NCX_Mj protein Na+ binding to outward-facing NCX_Mj. FIG
12 21 structure evidence (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
25 31 native protein_state (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
32 46 outward-facing protein_state (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
47 53 NCX_Mj protein (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
59 73 crystals grown experimental_method (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
84 87 Na+ chemical (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. FIG
36 39 Na+ chemical Colored spheres represent the bound Na+ (green) and water (red). FIG
52 57 water chemical Colored spheres represent the bound Na+ (green) and water (red). FIG
50 71 central binding sites site (b) Structural details and definition of the four central binding sites. FIG
4 20 electron density evidence The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
39 57 Fo-Fc ion omit map evidence The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
78 82 Smid site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
98 103 water chemical The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
130 134 Sext site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
136 139 SCa site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
144 148 Sint site The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
152 155 Na+ chemical The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres). FIG
89 92 Na+ chemical Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1). FIG
4 25 Fo – Fc ion-omit maps evidence All Fo – Fc ion-omit maps are calculated to 2.4 Å and contoured at 3σ for comparison. FIG
20 24 A206 residue_name_number The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG
39 42 Na+ chemical The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG
85 99 partially open protein_state The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG
107 115 occluded protein_state The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG
148 151 Na+ chemical The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively). FIG
9 12 Na+ chemical At 20 mM Na+, both conformations co-exist. FIG
75 80 water chemical No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG
101 104 SCa site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG
106 110 Sint site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG
115 119 Smid site No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. FIG
0 3 Na+ chemical Na+-occupancy dependent conformational change in NCX_Mj. FIG
49 55 NCX_Mj protein Na+-occupancy dependent conformational change in NCX_Mj. FIG
4 19 Superimposition experimental_method (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG
27 33 NCX_Mj protein (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG
34 52 crystal structures evidence (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG
128 131 Na+ chemical (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. FIG
25 34 interface site (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
43 46 TM6 structure_element (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
51 56 TM7ab structure_element (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
64 70 NCX_Mj protein (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
71 81 structures evidence (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
107 110 Na+ chemical (b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively). FIG
47 56 structure evidence Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG
60 63 low protein_state Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG
64 67 Na+ chemical Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. FIG
25 42 Na+-binding sites site (c) Close-up view of the Na+-binding sites. FIG
11 15 Sext site The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG
28 37 structure evidence The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG
41 44 low protein_state The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG
45 48 Na+ chemical The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. FIG
56 60 A206 residue_name_number Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG
78 89 coordinates bond_interaction Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG
90 93 Na+ chemical Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG
97 101 Sext site Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. FIG
47 64 ion binding sites site (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG
72 82 structures evidence (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG
86 90 high protein_state (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG
95 98 low protein_state (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG
100 103 Na+ chemical (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. FIG
9 25 solvent channels site Putative solvent channels are represented as light-purple surfaces. FIG
28 31 apo protein_state Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
32 41 structure evidence Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
45 51 NCX_Mj protein Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
66 70 Sr2+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
99 102 SCa site Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
106 123 crystals titrated experimental_method Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
135 139 Sr2+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
151 154 Na+ chemical Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
21 25 Sr2+ chemical Residues involved in Sr2+ coordination are labeled. FIG
98 107 Na+-bound protein_state There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state. FIG
0 3 T50 residue_name_number T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG
8 12 T209 residue_name_number T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG
30 40 coordinate bond_interaction T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG
41 45 Sr2+ chemical T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. FIG
5 8 Na+ chemical High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG
53 57 Sr2+ chemical High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG
70 76 NCX_Mj protein High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG
84 92 occluded protein_state High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). FIG
25 41 Fo – Fc omit map evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
49 58 structure evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
67 70 Na+ chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
128 135 density evidence The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
141 144 Na+ chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
154 157 H2O chemical The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O. FIG
4 8 Ca2+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
42 45 SCa site (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
49 53 Smid site (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
57 74 crystals titrated experimental_method (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
86 90 Ca2+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
102 105 Na+ chemical (b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2). FIG
60 75 Superimposition experimental_method The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
79 85 NCX_Mj protein The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
86 96 structures evidence The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
113 116 Na+ chemical The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
169 179 absence of protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
180 183 Na+ chemical The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
188 192 pH 4 protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
223 226 apo protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
259 276 ion-binding sites site The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
284 287 apo protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
292 299 high H+ protein_state The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state. FIG
19 22 E54 residue_name_number The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG
27 31 E213 residue_name_number The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG
41 48 low Na+ protein_state The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG
49 58 structure evidence The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. FIG
36 40 Sint site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
42 46 Smid site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
47 50 SCa site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
99 116 ion-binding sites site White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
120 123 apo protein_state White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
124 130 NCX_Mj protein White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. FIG
27 36 structure evidence Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG
40 56 outward-occluded protein_state Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG
58 76 fully Na+-occupied protein_state Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG
77 83 NCX_Mj protein Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG
132 135 Na+ chemical Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. FIG
19 29 simulation experimental_method (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
43 49 NCX_Mj protein (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
65 68 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
69 77 bound at protein_state (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
78 82 Sext site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
84 87 SCa site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
92 96 Sint site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
139 142 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
143 156 bound only at protein_state (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
157 160 SCa site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
165 169 Sint site (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
240 255 N-terminal half structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
259 262 TM7 structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
264 269 TM7ab structure_element (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
284 287 Na+ chemical (a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a). FIG
19 39 simulation snapshots evidence (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
61 64 Na+ chemical (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
65 73 bound at protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
74 77 SCa site (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
82 86 Sint site (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
125 132 without protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
137 140 Na+ chemical (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
141 146 bound protein_state (c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons). FIG
20 38 ion-binding region site (d) Close-up of the ion-binding region in the fully Na+-occupied state. FIG
46 64 fully Na+-occupied protein_state (d) Close-up of the ion-binding region in the fully Na+-occupied state. FIG
30 33 TM2 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG
35 38 TM3 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG
43 46 TM7 structure_element Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG
87 105 ion-binding region site Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG
113 135 partially Na+-occupied protein_state Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state. FIG
20 38 ion-binding region site (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG
46 54 Na+-free protein_state (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG
142 153 simulations experimental_method (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG
162 175 Na+-occupancy protein_state (f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f). FIG
63 101 Bias-Exchange Metadynamics simulations experimental_method These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods). FIG
4 29 Probability distributions evidence (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG
74 90 hydrogen-bonding bond_interaction (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG
102 107 TM7ab structure_element (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG
223 227 Sext site (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG
348 351 SCa site (g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1). FIG
108 111 NCX protein_type Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
117 165 Calculated conformational free-energy landscapes evidence Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
170 184 outward-facing protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
185 191 NCX_Mj protein Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
211 214 Na+ chemical Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
254 267 no ions bound protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound. FIG
4 15 free energy evidence The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG
106 122 aqueous channels site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG
138 142 Sext site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG
147 150 SCa site The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods). FIG
22 38 X-ray structures evidence Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
42 48 NCX_Mj protein Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
61 65 high protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
70 73 low protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
74 77 Na+ chemical Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
112 118 low pH protein_state Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study. FIG
4 23 Density isosurfaces evidence (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG
28 33 water chemical (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG
63 81 ion-binding region site (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG
120 153 conformational free-energy minima evidence (b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state. FIG
0 3 Na+ chemical Na+ ions are shown as green spheres. FIG
8 33 inverted-topology repeats structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG
41 52 transporter protein_type The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG
53 62 structure evidence The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG
111 116 TM1-5 structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG
126 132 TM6-10 structure_element The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). FIG
26 44 ion-binding region site (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG
57 90 conformational free-energy minima evidence (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG
25 28 Na+ chemical Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). FIG
33 38 water chemical Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). FIG
4 22 water-density maps evidence The water-density maps in (b) is shown here as a grey mesh. FIG
5 9 D240 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG
31 34 E54 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG
39 43 E213 residue_name_number Note D240 is protonated, while E54 and E213 are ionized. FIG
108 111 NCX protein_type Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
117 150 Calculated free-energy landscapes evidence Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
155 169 outward-facing protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
170 176 NCX_Mj protein Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
186 190 Ca2+ chemical Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
199 215 fully protonated protein_state Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state. FIG
4 15 free energy evidence The free energy is plotted as in Fig. 5. FIG
4 8 Ca2+ chemical For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG
12 15 map evidence For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG
144 147 map evidence For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6). FIG
16 19 map evidence The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
39 43 open protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
66 75 semi-open protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
80 88 occluded protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
166 174 bound to protein_state The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
218 221 Na+ chemical The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+). FIG
22 40 crystal structures evidence Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG
58 62 Ca2+ chemical Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG
84 90 low pH protein_state Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG
95 102 high H+ protein_state Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. FIG
4 29 Water-density isosurfaces evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG
106 124 free-energy minima evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG
132 148 free-energy maps evidence (b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps. FIG
4 8 Ca2+ chemical The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG
100 118 ion-binding region site The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG
131 164 conformational free-energy minima evidence The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima. FIG
25 29 Ca2+ chemical Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). FIG
34 39 water chemical Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). FIG
4 22 water-density maps evidence The water-density maps in (b) are shown here as a grey mesh. FIG
7 15 occluded protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
27 31 Ca2+ chemical In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
32 37 bound protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
39 44 helix structure_element In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
45 50 TM7ab structure_element In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
83 97 fully occupied protein_state In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
98 101 Na+ chemical In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
128 134 Ala206 residue_name_number In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
143 171 hydrogen-bonding interaction bond_interaction In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
177 183 Ser210 residue_name_number In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210. FIG
62 65 NCX protein_type Structural mechanism of extracellular forward ion exchange in NCX. FIG
23 28 Ala47 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
33 37 TM2b structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
43 49 Ala206 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
54 58 TM7b structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
84 89 Glu54 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
94 98 TM2c structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
104 110 Glu213 residue_name_number The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
115 119 TM7c structure_element The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes. FIG
39 53 gating helices structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG
54 57 TM1 structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG
62 65 TM6 structure_element The green open cylinders represent the gating helices TM1 and TM6. FIG
32 50 crystal structures evidence Asterisks mark the states whose crystal structures have been determined in this study. FIG
65 98 calculated free-energy landscapes evidence These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG
120 131 Ca2+-loaded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG
132 146 outward-facing protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG
147 155 occluded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG
170 178 unloaded protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG
180 190 fully open protein_state These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state. FIG