BACKGROUND
the s <dig> calcium-binding protein family of vertebrate, metal-regulated proteins plays pivotal roles in a wide variety of intracellular and extracellular functions including cell growth, inflammation, membrane remodeling and chemotaxis  <cit>  and are implicated in many diseases including cancer  <cit> .
hallmarks of the s <dig> protein family include their small size  and the presence of a canonical  ef-hand motif and a non-canonical  ef-hand motif  <cit> .
additional complexity in s <dig> proteins is derived from their ability to adopt a non-covalent anti-parallel homo/heterodimers that can further associate to yield higher order multimers such as tetramers and hexamers  <cit> .
while most s <dig> proteins share moderate sequence identity, s100a <dig> and s100a <dig> are 93% identical in sequence yet intriguingly display divergent functions.
s100a <dig>  for example, is implicated in inflammatory skin diseases and in several cancers including breast cancer  <cit>  where it is associated with aggressive estrogen receptor negative tumors and poor prognosis  <cit> .
intracellular s100a <dig> has also been shown to interact with the multifunctional signaling protein jab <dig>  resulting in translocation of jab <dig> to the nucleus where it mediates s100a <dig> tumorigenic effects  <cit> .
while s100a <dig> is also highly expressed in psoriatic lesions  <cit> , it displays distinct localizations in skin and breast, divergent functions in epithelial maturation and skin inflammation  <cit>  and it is currently unknown whether s100a <dig> contributes to breast cancer progression  <cit> .
s100a <dig> and s100a <dig> are also capable of binding different surface receptors  <cit> .
s100a <dig> interacts with rage  to mediate chemotaxis of leukocytes  <cit>  while s100a <dig> appears to mediate chemotaxis through an unidentified g protein coupled receptor  <cit> .
the ability of many s <dig> proteins to coordinate zinc is central to their function  <cit> .
for example, the ability of s100a <dig> to engage rage and exert antimicrobial effects are both dependent on zinc  <cit>  <cit> .
these observations suggest a regulatory role although the zinc coordination geometry and affinity suggest a structural role  <cit> .
it is not yet known whether zinc binding plays a structural or regulatory role in s100a <dig> and s100a <dig> function.
to our knowledge, there is no data to suggest s100a <dig> function is zinc-dependent.
thus far, two types of s100-family zinc sites have been proposed  <cit> .
in the first type , the zinc is coordinated by  <dig> histidines and an aspartate  or with four histidines .
the second type  is proposed to contain primarily cysteines as the zinc-coordinating residues though no structure of an s <dig> ‘cys-zn’ binding site has been reported.
ultimately defining the zinc binding capacity for s100a <dig> is crucial to understanding its function and appropriately categorizing it within the s <dig> family of proteins.
to this end, we report the structural characterization of s100a <dig> and an asp24gly variant of s100a <dig> to  <dig>  Å and  <dig>  Å, respectively.
these data reveal an unexpected structural compensation to retain zinc binding in s100a <dig> and a structural rationale for the inability of s100a <dig> to coordinate rage and the prediction that s100a <dig> will not bind jab <dig>
methods
protein production and purification
s100a <dig>  and s100a <dig> d24g were generated using using quikchange mutagenesis  of wildtype s100a <dig>  <cit>  and recombinantly produced as a thioredoxin fusion with an n-terminal hexa histidine tag.
protein production was performed in e. coli rosetta-gammi b in overnight express™ instant tb medium  overnight at 37°c.
the cells were harvested, resuspended in binding buffer , lysed using a french press, applied to a his-trap column and eluted using an increasing gradient of imidazole to 500 mm.
fractions were analyzed with sds-page, pooled based on purity, concentrated and the histidine tag removed by thrombin digestion prior to a final purification step using superdex sx <dig> gel filtration column.
due to the absence of tryptophan or phenylalanine residues, protein concentration was calculated based on amino acid analysis.
crystallization, data collection, processing and structure solution
crystallization was performed at 18 °c using the sitting drop vapour diffusion method.
s100a <dig> was crystallized at 26 mg/ml in 45–50 mm zinc acetate dihydrate, 18-20% peg  <dig> and s100a7d24g was crystallized at 19 mg/ml in  <dig>  m ammonium sulfate,  <dig>  m bistris ph  <dig> , 20-25% peg  <dig>  diffraction data were collected on beamline 9– <dig> at ssrl and processed with d*trek  <cit> .
data collection and refinement statistics are presented in table  <dig>  refinement was performed with the ccp <dig> suite of programs  <cit> .
the initial phases were obtained by molecular replacement  using molrep  <cit>  with the monomeric form of native s100a <dig>  solvent atoms were selected using coot  <cit>  and the structure refined with refmac  <cit> .
stereo-chemical analysis of the refined structures was performed with procheck and sfcheck in ccp <dig>  <cit> .
overall 5% of the reflections were set aside for calculation of rfree.
protein data bank accession number
coordinates and structure factor accession codes are as follows: s100a <dig>  and s100a7d24g .
RESULTS
s100a <dig> retains the ability to coordinate zinc
to investigate the differences in zinc binding between s100a <dig> and s100a <dig>  we recombinantly produced human s100a <dig> in e. coli and purified the protein to homogeneity using nickel affinity chromatography.
similar to s100a <dig>  s100a <dig> eluted as a stable dimer from a size exclusion column  indicating that the seven divergent residues   did not disrupt the multimeric structure.
s100a <dig> crystallized as a monomer in the asymmetric unit of the monoclinic cell with the dimer generated by crystallographic symmetry .
molecular replacement was used to solved the s100a <dig> structure with native s100a <dig>  <cit>  as the search model and all waters, zinc and calcium ions removed and the  <dig> divergent residues  mutated to alanine.
the s100a <dig> structure was refined to a resolution of  <dig>  Å and displayed an overall root mean squared deviation  of  <dig>  Å <dig> over  <dig> cα atoms relative to s100a <dig>
during the initial refinement stages of the s100a <dig> structure an  <dig> sigma difference electron density peak was observed that overlaid with the bound zinc in s100a <dig> .
accordingly, a zinc ion was modeled and refined at full occupancy resulting in a b-factor of  <dig>  Å <dig>  thus, it was apparent that substitution of gly <dig> in s100a <dig> for the zinc ligand asp <dig>  did not abrogate zinc binding.
remarkably, the coordination geometry of the three histidine ligands  and his <dig> - monomer 1)  is virtually unchanged between s100a <dig>  and s100a <dig> .
of these ligands, only his <dig> displayed a measurable shift with a 15° reorientation and a shift of the imidazole ring ne <dig> and nd <dig> by  <dig>  Å and  <dig>  Å, respectively, likely due to the position of his <dig> on a mobile surface loop .
to complete the distorted tetrahedral geometry of the bound zinc in s100a <dig>  a chloride ion was modeled, which refined to a distance of  <dig>  Å from the zinc and a b factor of  <dig>  Å <dig>  while water molecules are commonly observed as labile ligands in zinc coordination sites, we elected to model a chloride ion using the b factor as a guide and based on the fact that chloride was the dominant anion in our buffer.
while asp <dig> has been previously observed as a bindentate ligand  <cit> , we measured zn-o bond distance of  <dig>  Å  and  <dig>  Å .
the shorter bond length between zn and asp <dig> od <dig> suggests that s100a <dig> asp  <dig> likely acts as a monodentate ligand, and that both s100a <dig> and s100a <dig> bind zinc in a  <dig> coordinate geometry.
in support of the relevance of the observed zinc coordination by s100a <dig>  we obtained preliminary isothermal titration calorimetry data by 1) treating with chelex resin to remove zinc and 2) back-titrating with zncl <dig>  to allow direct measurement of binding.
these preliminary results show nearly identical dh values of − <dig>  ±  <dig>  kcal/mol and − <dig>  ±  <dig>  kcal/mol for s100a <dig> and s100a <dig>  respectively, suggesting similar strengths of zn coordination by both proteins in spite of the change of coordination environment.
zn affinities determined by itc  vary depending on buffer composition, but are generally within five-fold of each other under a given set of conditions.
the construction of pseudopolarograms on s100a <dig> and s100a <dig> samples using anodic stripping voltammetry, and the analysis of resulting half-wave potential shifts also indicate that both proteins bind zinc with similar affinity  <cit> .
full solution-phase data will be reported in due course.
replacement of asp <dig>  with a glycine  is sufficient to produce a zinc-binding site unique among s <dig> proteins
to confirm that the substitution of asp <dig> with a glycine was solely responsible for the unique zinc-binding site observed in s100a <dig>  we generated the d24g variant of s100a <dig>  s100a7d24g eluted as a dimer from the sec, and crystallized with one monomer in the asymmetric unit of the tetragonal cell.
diffraction data was collected to a resolution of  <dig>  Å  and the structure solved by molecular replacement.
the s100a7d24g and s100a <dig> structures aligned closely with an r.m.s.d of  <dig>  Å over  <dig> cα atoms.
subtle adjustments were observed in the orientation of the histidine ligands, due to differential packing of surface loops between the monoclinic and tetragonal crystal forms.
to validate the modeling of zinc at the dimer interface, we calculated an anomalous difference map using data collected at the zinc edge of  <dig>  nm.
a large anomalous difference peak  was observed where the zinc had been modeled confirming the identity of the bound metal as zinc .
while crystallization of s100a <dig> was completed in the presence of zinc, s100a7d24g was crystallized in the absence of zinc, and yet zinc binding was observed in the same geometry.
these observations indicate that the zinc-binding observed in the s100a <dig> is not an artifact.
similar to s100a <dig>  a chloride ion was modeled as the exogenous fourth ligand to complete the distorted tetrahedral geometry.
the incorporation of a dissociable ligand such as chloride may enable a binding partner to donate a fourth ligand thereby ensuring a tighter ligand-receptor complex.
a second zinc atom was observed in the s100a <dig> structure , which was not observed in the s100a7d24g mutant.
we expect this is an artifact as s100a <dig> wt was crystallized in high concentrations  of zinc while s100a7d24g was crystallized in the absence of zinc.
analysis of zinc binding sites between s100a <dig>  s100a <dig>  s100b and s100a <dig> reveal highly conserved coordination geometry for the metal positioned at the dimer interface.
while sequence analysis shows that s100a <dig> and s100b encode a glycine at the equivalent sequence  position to  <dig> in s100a <dig>  a three-residue insertion  results in a histidine  or an aspartate  being correctly positioned to serve as the fourth zinc ligand .
thus, it appears that multiple different strategies are used by s <dig> proteins to maintain the distorted coordination geometry of the zinc bound at the dimer interface consistent with a central functional role.
in fact, the strict conservation of the zinc site is remarkable especially in the context of structural divergences relative to s100a <dig>  with substantial reorganization in nearby surface loops .
it is not clear why the zinc binding affinities of rat s100b  and human s100a <dig>  are much higher than human s100a <dig>   <cit> .
altered surface chemistry on s100a <dig> may explain the lack of rage and predicted jab <dig> binding
the second cluster of amino acid differences  between s100a <dig> and s100a <dig> is localized to an ordered loop between helices iib and iii and extending into the n-terminal portion of helix iii.
the center of this cluster is positioned approximately 24 Å from the closest zinc-binding site.
intriguingly, this region in s100a <dig> has been implicated in mediating interaction with jab <dig>  <cit>  and between s100b and rage  <cit> .
the inability of s100a <dig> to bind rage  <cit> , and the prediction that s100a <dig> lacks a functional jab <dig> binding site  <cit> , suggests that this localized cluster of residues is a hot spot surface for ligand-receptor interactions in the s <dig> family of proteins.
functional consequences based on altered secondary structure have been observed in s100a <dig> where mutation of asp <dig> to gly resulted in shortening of helix iii  <cit> .
however, no such changes were observed in s100a <dig> and overlay of helix iii  of s100a <dig> and s100a <dig> yielded an r.m.s.d of only  <dig>  Å over  <dig> cα atoms.
overall, the replacement of asp <dig> with thr and asn <dig> with his substantially alters the surface chemistry and may explain the inability of s100a <dig> to bind rage.
despite being capable of specific interactions with unique targets, the emerging data on s100-rage interactions suggest that s <dig> proteins have evolved to also interact with a common receptor, rage, via unique structural binding sites and in distinct multimeric states  <cit> .
adding to the combinatorial possibilities of s100-rage interactions is the observation that the rage is capable of multimerization: s100b tetramers interact with rage dimers, s100b dimers interact with rage monomers  <cit> , and s100a <dig> hexamers interact with rage tetramers  <cit> .
however, close examination of the amino acid sequences and structures of s100b, s100a <dig>  s100a <dig> and s100a <dig>  offers little to explain why s100a <dig> does not bind rage while other s <dig> proteins do.
extensive scanning mutagenesis or structural characterization of the complexes will ultimately be required to define the core features that promote macromolecular assembly.
CONCLUSIONS
zinc-coordination is a dynamic process and can play a role in the regulation of protein function  <cit> .
recent studies have suggested that misregulation of zinc homeostasis is associated with cancer  <cit> .
distinct metal binding and protein-interacting functions have been described for s100a <dig> and s100a <dig> despite their 93% sequence identity.
zinc is required for several s100a <dig> functions, including antimicrobial action and rage binding.
s100a <dig> does not bind rage and a zinc-dependent function has not yet been identified, which has led to the suggestion that s100a <dig> does not bind zinc.
here we demonstrate that s100a <dig> does in fact coordinate zinc in a distorted tetrahedral geometry with an exogenous ligand that is unique to s <dig> proteins.
these data place s100a <dig> as the first member of what is likely to be a growing subgroup of s <dig> proteins.
further studies are required to establish the mechanism of receptor interactions with the s <dig> proteins, but divergent solutions to maintain zinc binding is undoubtedly going to play a central role.
competing interests
the authors declare that they have no competing interests.
author’s contributions
jm, fh and mb conceived of the study and along with mt, aw and jc designed the experiments.
jm and mb performed the crystallization, data collection and structure solution.
jm, mt and bf produced the recombinant s <dig> proteins.
aw and jc performed the solution binding studies.
jm and mb wrote the manuscript with editing contributed by mt, fp and fh.
all authors read and approved the final manuscript.
acknowledgments
this work was supported by a research grant from the canadian institutes of health research  to mb  and the canadian breast cancer foundation, bc/yukon region to fh.
mlt is supported by a julie payette natural science and engineering research council  fellowship.
alw is supported by an nserc pgsd and michael smith foundation for health research  fellowship.
mb and fh are msfhr scholars and tier ii canada research chairs.
the authors gratefully acknowledge the beamline support staff at the stanford synchrotron radition laboratory .

 the s calcium-binding protein family of vertebrate, metal-regulated proteins plays pivotal roles in a wide variety of intracellular and extracellular functions including cell growth, inflammation, membrane remodeling and chemotaxis and are implicated in many diseases including cancer. hallmarks of the s protein family include their small size and the presence of a canonical ef-hand motif and a non-canonical ef-hand motif. additional complexity in s proteins is derived from their ability to adopt a non-covalent anti-parallel homo/heterodimers that can further associate to yield higher order multimers such as tetramers and hexamers. while most s proteins share moderate sequence identity, s100a and s100a are 93% identical in sequence yet intriguingly display divergent functions. s100a for example, is implicated in inflammatory skin diseases and in several cancers including breast cancer where it is associated with aggressive estrogen receptor negative tumors and poor prognosis. intracellular s100a has also been shown to interact with the multifunctional signaling protein jab resulting in translocation of jab to the nucleus where it mediates s100a tumorigenic effects. while s100a is also highly expressed in psoriatic lesions, it displays distinct localizations in skin and breast, divergent functions in epithelial maturation and skin inflammation and it is currently unknown whether s100a contributes to breast cancer progression. s100a and s100a are also capable of binding different surface receptors. s100a interacts with rage to mediate chemotaxis of leukocytes while s100a appears to mediate chemotaxis through an unidentified g protein coupled receptor. the ability of many s proteins to coordinate zinc is central to their function. for example, the ability of s100a to engage rage and exert antimicrobial effects are both dependent on zinc. these observations suggest a regulatory role although the zinc coordination geometry and affinity suggest a structural role. it is not yet known whether zinc binding plays a structural or regulatory role in s100a and s100a function. to our knowledge, there is no data to suggest s100a function is zinc-dependent. thus far, two types of s100-family zinc sites have been proposed. in the first type, the zinc is coordinated by histidines and an aspartate or with four histidines. the second type is proposed to contain primarily cysteines as the zinc-coordinating residues though no structure of an s ‘cys-zn’ binding site has been reported. ultimately defining the zinc binding capacity for s100a is crucial to understanding its function and appropriately categorizing it within the s family of proteins. to this end, we report the structural characterization of s100a and an asp24gly variant of s100a to  Å and  Å, respectively. these data reveal an unexpected structural compensation to retain zinc binding in s100a and a structural rationale for the inability of s100a to coordinate rage and the prediction that s100a will not bind jab methods protein production and purification s100a and s100a d24g were generated using using quikchange mutagenesis of wildtype s100a and recombinantly produced as a thioredoxin fusion with an n-terminal hexa histidine tag. protein production was performed in e. coli rosetta-gammi b in overnight express™ instant tb medium overnight at 37°c. the cells were harvested, resuspended in binding buffer, lysed using a french press, applied to a his-trap column and eluted using an increasing gradient of imidazole to 500 mm. fractions were analyzed with sds-page, pooled based on purity, concentrated and the histidine tag removed by thrombin digestion prior to a final purification step using superdex sx gel filtration column. due to the absence of tryptophan or phenylalanine residues, protein concentration was calculated based on amino acid analysis. crystallization, data collection, processing and structure solution crystallization was performed at 18 °c using the sitting drop vapour diffusion method. s100a was crystallized at 26 mg/ml in 45–50 mm zinc acetate dihydrate, 18-20% peg and s100a7d24g was crystallized at 19 mg/ml in  m ammonium sulfate,  m bistris ph, 20-25% peg diffraction data were collected on beamline 9– at ssrl and processed with d*trek. data collection and refinement statistics are presented in table  refinement was performed with the ccp suite of programs. the initial phases were obtained by molecular replacement using molrep with the monomeric form of native s100a solvent atoms were selected using coot and the structure refined with refmac. stereo-chemical analysis of the refined structures was performed with procheck and sfcheck in ccp. overall 5% of the reflections were set aside for calculation of rfree. protein data bank accession number coordinates and structure factor accession codes are as follows: s100a and s100a7d24g.  s100a retains the ability to coordinate zinc to investigate the differences in zinc binding between s100a and s100a we recombinantly produced human s100a in e. coli and purified the protein to homogeneity using nickel affinity chromatography. similar to s100a s100a eluted as a stable dimer from a size exclusion column indicating that the seven divergent residues did not disrupt the multimeric structure. s100a crystallized as a monomer in the asymmetric unit of the monoclinic cell with the dimer generated by crystallographic symmetry. molecular replacement was used to solved the s100a structure with native s100a as the search model and all waters, zinc and calcium ions removed and the divergent residues mutated to alanine. the s100a structure was refined to a resolution of  Å and displayed an overall root mean squared deviation of  Å over cα atoms relative to s100a during the initial refinement stages of the s100a structure an sigma difference electron density peak was observed that overlaid with the bound zinc in s100a. accordingly, a zinc ion was modeled and refined at full occupancy resulting in a b-factor of  Å thus, it was apparent that substitution of gly in s100a for the zinc ligand asp did not abrogate zinc binding. remarkably, the coordination geometry of the three histidine ligands and his - monomer 1) is virtually unchanged between s100a and s100a. of these ligands, only his displayed a measurable shift with a 15° reorientation and a shift of the imidazole ring ne and nd by  Å and  Å, respectively, likely due to the position of his on a mobile surface loop. to complete the distorted tetrahedral geometry of the bound zinc in s100a a chloride ion was modeled, which refined to a distance of  Å from the zinc and a b factor of  Å while water molecules are commonly observed as labile ligands in zinc coordination sites, we elected to model a chloride ion using the b factor as a guide and based on the fact that chloride was the dominant anion in our buffer. while asp has been previously observed as a bindentate ligand, we measured zn-o bond distance of  Å and  Å. the shorter bond length between zn and asp od suggests that s100a asp likely acts as a monodentate ligand, and that both s100a and s100a bind zinc in a coordinate geometry. in support of the relevance of the observed zinc coordination by s100a we obtained preliminary isothermal titration calorimetry data by 1) treating with chelex resin to remove zinc and 2) back-titrating with zncl to allow direct measurement of binding. these preliminary results show nearly identical dh values of −  ±   kcal/mol and −  ±   kcal/mol for s100a and s100a respectively, suggesting similar strengths of zn coordination by both proteins in spite of the change of coordination environment. zn affinities determined by itc vary depending on buffer composition, but are generally within five-fold of each other under a given set of conditions. the construction of pseudopolarograms on s100a and s100a samples using anodic stripping voltammetry, and the analysis of resulting half-wave potential shifts also indicate that both proteins bind zinc with similar affinity. full solution-phase data will be reported in due course. replacement of asp with a glycine is sufficient to produce a zinc-binding site unique among s proteins to confirm that the substitution of asp with a glycine was solely responsible for the unique zinc-binding site observed in s100a we generated the d24g variant of s100a s100a7d24g eluted as a dimer from the sec, and crystallized with one monomer in the asymmetric unit of the tetragonal cell. diffraction data was collected to a resolution of  Å and the structure solved by molecular replacement. the s100a7d24g and s100a structures aligned closely with an r.m.s.d of  Å over cα atoms. subtle adjustments were observed in the orientation of the histidine ligands, due to differential packing of surface loops between the monoclinic and tetragonal crystal forms. to validate the modeling of zinc at the dimer interface, we calculated an anomalous difference map using data collected at the zinc edge of  nm. a large anomalous difference peak was observed where the zinc had been modeled confirming the identity of the bound metal as zinc. while crystallization of s100a was completed in the presence of zinc, s100a7d24g was crystallized in the absence of zinc, and yet zinc binding was observed in the same geometry. these observations indicate that the zinc-binding observed in the s100a is not an artifact. similar to s100a a chloride ion was modeled as the exogenous fourth ligand to complete the distorted tetrahedral geometry. the incorporation of a dissociable ligand such as chloride may enable a binding partner to donate a fourth ligand thereby ensuring a tighter ligand-receptor complex. a second zinc atom was observed in the s100a structure, which was not observed in the s100a7d24g mutant. we expect this is an artifact as s100a wt was crystallized in high concentrations of zinc while s100a7d24g was crystallized in the absence of zinc. analysis of zinc binding sites between s100a s100a s100b and s100a reveal highly conserved coordination geometry for the metal positioned at the dimer interface. while sequence analysis shows that s100a and s100b encode a glycine at the equivalent sequence position to in s100a a three-residue insertion results in a histidine or an aspartate being correctly positioned to serve as the fourth zinc ligand. thus, it appears that multiple different strategies are used by s proteins to maintain the distorted coordination geometry of the zinc bound at the dimer interface consistent with a central functional role. in fact, the strict conservation of the zinc site is remarkable especially in the context of structural divergences relative to s100a with substantial reorganization in nearby surface loops. it is not clear why the zinc binding affinities of rat s100b and human s100a are much higher than human s100a. altered surface chemistry on s100a may explain the lack of rage and predicted jab binding the second cluster of amino acid differences between s100a and s100a is localized to an ordered loop between helices iib and iii and extending into the n-terminal portion of helix iii. the center of this cluster is positioned approximately 24 Å from the closest zinc-binding site. intriguingly, this region in s100a has been implicated in mediating interaction with jab and between s100b and rage. the inability of s100a to bind rage, and the prediction that s100a lacks a functional jab binding site, suggests that this localized cluster of residues is a hot spot surface for ligand-receptor interactions in the s family of proteins. functional consequences based on altered secondary structure have been observed in s100a where mutation of asp to gly resulted in shortening of helix iii. however, no such changes were observed in s100a and overlay of helix iii of s100a and s100a yielded an r.m.s.d of only  Å over cα atoms. overall, the replacement of asp with thr and asn with his substantially alters the surface chemistry and may explain the inability of s100a to bind rage. despite being capable of specific interactions with unique targets, the emerging data on s100-rage interactions suggest that s proteins have evolved to also interact with a common receptor, rage, via unique structural binding sites and in distinct multimeric states. adding to the combinatorial possibilities of s100-rage interactions is the observation that the rage is capable of multimerization: s100b tetramers interact with rage dimers, s100b dimers interact with rage monomers, and s100a hexamers interact with rage tetramers. however, close examination of the amino acid sequences and structures of s100b, s100a s100a and s100a offers little to explain why s100a does not bind rage while other s proteins do. extensive scanning mutagenesis or structural characterization of the complexes will ultimately be required to define the core features that promote macromolecular assembly.  zinc-coordination is a dynamic process and can play a role in the regulation of protein function. recent studies have suggested that misregulation of zinc homeostasis is associated with cancer. distinct metal binding and protein-interacting functions have been described for s100a and s100a despite their 93% sequence identity. zinc is required for several s100a functions, including antimicrobial action and rage binding. s100a does not bind rage and a zinc-dependent function has not yet been identified, which has led to the suggestion that s100a does not bind zinc. here we demonstrate that s100a does in fact coordinate zinc in a distorted tetrahedral geometry with an exogenous ligand that is unique to s proteins. these data place s100a as the first member of what is likely to be a growing subgroup of s proteins. further studies are required to establish the mechanism of receptor interactions with the s proteins, but divergent solutions to maintain zinc binding is undoubtedly going to play a central role. competing interests the authors declare that they have no competing interests. author’s contributions jm, fh and mb conceived of the study and along with mt, aw and jc designed the experiments. jm and mb performed the crystallization, data collection and structure solution. jm, mt and bf produced the recombinant s proteins. aw and jc performed the solution binding studies. jm and mb wrote the manuscript with editing contributed by mt, fp and fh. all authors read and approved the final manuscript. acknowledgments this work was supported by a research grant from the canadian institutes of health research to mb and the canadian breast cancer foundation, bc/yukon region to fh. mlt is supported by a julie payette natural science and engineering research council fellowship. alw is supported by an nserc pgsd and michael smith foundation for health research fellowship. mb and fh are msfhr scholars and tier ii canada research chairs. the authors gratefully acknowledge the beamline support staff at the stanford synchrotron radition laboratory.
remarkably, despite replacement of the asp ligand, zinc binding is retained at the s100a dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. of the seven divergent residues that differentiate s100a and s100a four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface. while the majority of human s proteins share 25-65% sequence similarity, s100a and its recently identified paralog, s100a display 93% sequence identity. to investigate the structural and functional consequences of these divergent clusters, we report the x-ray crystal structures of s100a and s100a7d24g, a hybrid variant where the zinc ligand asp of s100a has been substituted with the glycine of s100a to  Å and  Å resolution, respectively. collectively, these data provide novel insight into the structural and functional consequences of the divergent surfaces between s100a and s100a that may be exploited for targeted therapies. the altered surface chemistry between s100a and s100a that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct protein targets. here we demonstrate that s100a retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among s proteins.
 s proteins are a family of small, ef-hand containing calcium-binding signaling proteins that are implicated in many cancers. while the majority of human s proteins share 25-65% sequence similarity, s100a and its recently identified paralog, s100a display 93% sequence identity. intriguingly, however, s100a and s100a serve distinct roles in inflammatory skin disease; s100a signals through the receptor for advanced glycation products in a zinc-dependent manner, while s100a signals through a yet unidentified g-protein coupled receptor in a zinc-independent manner. of the seven divergent residues that differentiate s100a and s100a four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface.  to investigate the structural and functional consequences of these divergent clusters, we report the x-ray crystal structures of s100a and s100a7d24g, a hybrid variant where the zinc ligand asp of s100a has been substituted with the glycine of s100a to  Å and  Å resolution, respectively. remarkably, despite replacement of the asp ligand, zinc binding is retained at the s100a dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for s100a and s100a additionally, the predicted receptor-binding surface on s100a is substantially more basic in s100a without incurring structural rearrangement.  here we demonstrate that s100a retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among s proteins. the altered surface chemistry between s100a and s100a that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct protein targets. collectively, these data provide novel insight into the structural and functional consequences of the divergent surfaces between s100a and s100a that may be exploited for targeted therapies. s100a15s100a7zinc-bindingef handx-ray crystallography 