BACKGROUND
s <dig> proteins belong to the ef-hand calcium-binding protein superfamily and count more than  <dig> members that are expressed exclusively in vertebrates, in a tissue- and cell-specific manner  <cit> .
under homeostatic conditions, they are found in the cytoplasm or in the nucleus of eukaryotic cells where they regulate vital processes, generally in a calcium-dependent manner  <cit> .
s <dig> protein expression is often upregulated during inflammation.
in addition, these proteins can be secreted or passively released in the extracellular matrix where they become damage-associated molecular patterns  and exert cytokine-like functions, through specific membrane-bound receptors , thus promoting sustained inflammation and tissue damage in various pathological settings associated with cardiovascular complications, neurodegenerative disorders and cancers .
as a consequence, s <dig> proteins are considered both as valuable biological markers and important therapeutical targets for various inflammatory conditions  <cit> .
s100a <dig> and s100a <dig> constitute two of the most potent pro-inflammatory molecules among the s <dig> protein family  <cit> .
they are constitutively expressed by leukocytes of myeloid lineage  <cit> .
although products of distinct genes, the two proteins are often co-expressed and account for up to 45 % of the total cytosolic protein pool in human neutrophils  <cit> .
as for all their congeners, s100a <dig> and s100a <dig> form homodimers as a minimal, active unit  <cit> .
however, they usually exert their physiological function as a s100a8/a <dig> heterodimer, also termed calprotectin, and they can further associate via heterotetramerization  <cit> .
the s100a8/a <dig> heterodimer is proposed to regulate various homeostatic processes including fatty acid transport, cytoskeleton reorganization and myeloid cell differentiation .
s100a <dig> and s100a <dig> have no signal sequences for classical secretion, but can be released from activated leukocytes, either by active secretion via a tubulin-dependent pathway, or by passive release, within neutrophil extracellular traps  or from necrotic cells .
as for all s <dig> proteins, s100a <dig>  s100a <dig>  and their heterodimer bind two calcium ions per monomer, one in each ef-hand motif  <cit> .
in addition to calcium, calprotectin can bind zn2+, mn2+, cu2+, and fe2+ and, by efficiently sequestrating these divalent cations in the extracellular environment, it acts as a potent antimicrobial agent .
interestingly, this antibacterial property is restricted to the s100a8/a <dig> heterodimer  <cit>  although both homodimers are expected to bind zinc as well, based on sequence comparisons  <cit> .
indeed, zinc binding has been reported for several members of the s <dig> protein family  <cit>  and the determination of the three-dimensional structures of both s100b, s100a <dig>  s100a <dig> and s100a <dig> complexed to zinc has revealed two common, symmetrical zinc binding sites at the dimer interface formed by highly conserved histidines and possibly one or two aspartate/glutamate .
these zn2+-coordinating residues are conserved in s100a <dig> and s100a <dig>  suggesting that both proteins could bind zinc in a similar fashion.
however, no structural data are yet available to precisely characterize the zinc binding mode of either s100a <dig> and s100a <dig> homodimers or s100a8/a <dig> heterodimer.
zinc was furthermore shown to enhance the oligomerization of these proteins, either as homo- and heterotetramers or as much more complex amyloid structures  <cit> .
finally, it has been proposed that the activity of extracellular s <dig> proteins, including s100a <dig> and s100a <dig>  would be regulated by other divalent cations than calcium, notably zn2+, since the two ef-hand ca2+-binding sites would be fully saturated at all time in the extracellular compartment due to the high calcium concentrations  <cit> .
zinc appears therefore as an important regulator of the structural organization and the resulting functional properties of both s100a <dig> and s100a <dig>  however, no structural information is yet available to decipher the intimate mechanisms by which this regulation is achieved.
to better understand how zinc modulates the structure-function relationship of s100a <dig> and s100a <dig>  we have undertaken the structural studies of these s <dig> proteins in the presence of zinc.
here we report the crystallographic structures of human s100a <dig>  bound to zinc and calcium at  <dig>  and  <dig>  Å, respectively.
the first structure reveals a classical, all-histidine zinc-binding motif, thereby classifying s100a <dig> as a genuine his-zn s <dig> protein.
the second structure displays an atypical 2-cacodylate complex in one of the two zn-sites per homodimer, leading to ligand swapping and creation of two novel zn-sites.
analysis of the quaternary arrangement of hs100a <dig> in both crystal forms suggests that zinc enhances tetramer stabilization and could further participate in the formation of higher oligomeric species and/or in the interaction with s100a <dig> binding partners.
RESULTS
a classical his-zn binding motif
in the presence of both zinc and calcium, hs100a <dig> gave rise to two new crystal forms displaying a p <dig> and a c <dig> symmetry, as compared to ca2+-bound hs100a <dig> which crystallized in p <dig>  <cit>  .
both structures could nevertheless be solved by molecular replacement using the previously reported ca2+-hs100a <dig> homodimer structure.
for crystal form  <dig> , structure determination was also successful using sad-phasing in phenix.autosolve  <cit>  from the dataset collected at the zn peak  .
in that case,  <dig> sites were identified corresponding to the eight zn2+ ions and four of the eight ca2+ ions .
the overall figures of merit after sad-phasing and after density modification using resolve were  <dig>  and  <dig> , respectively .
in any case, the position of the zinc ions could be unambiguously identified in both structures from anomalous difference fourier maps calculated using phases and figure of merit weights from the best refined atomic model containing no ions obtained with the native dataset and anomalous differences from the datasets collected at wavelengths of  <dig>  and  <dig>  Å .table  <dig> data collection and refinement statistics
i/σ
r
meas
values for the outer shell are given in parentheses
fig.
<dig> the crystallographic structure of zn2+/ca2+-bound hs100a <dig>  a anomalous difference fourier maps calculated using phases and weight from the best refined atomic model  obtained with the native dataset  and anomalous differences from the datasets collected at wavelengths of  <dig>  Å  and  <dig>  Å .
the final model displayed as cartoon in beige and purple is superimposed for comparison.
the anomalous signal clearly disappears for the wavelength above the zn maximal absorbance peak  as judged by the fact that almost no density is visible for the second anomalous difference fourier map .
b overview of the structure of zn2+/ca2+-bound hs100a <dig> derived from crystal form  <dig> at  <dig>  Å resolution showing two zinc bound at the homodimer interface .
the four calcium ions are displayed as green spheres.
c close-up view on the zn2+-binding site revealing an all-his binding motif formed by two residues from each monomer.
d in two out of eight molecules within the asymmetric unit, his <dig> from monomer a is replaced by a chloride ion
the overall structure of zn2+/ca2+-bound hs100a <dig> from crystal form  <dig> is displayed in fig.
1b.
the protein arranges in a canonical homodimer that contains four calcium ions, one in each ef-hand, and two zinc ions at the interface between the two monomers .
although zinc can replace calcium in the ef-hand motifs when present alone  <cit> , only ca2+ was found in all ef-hands of our model as judged from the anomalous difference fourier maps .
the two symmetrical zn-sites are formed by two residues from each monomer: from the first monomer, his <dig> in helix h <dig> and his <dig> in the first ef-hand, and from the second monomer, his <dig> and his <dig> at the c-terminus of helix h <dig> .
his <dig> appears however somewhat flexible and in two out of the eight molecules present in the asymmetric unit of our structure, it is flipped away from the zn-site by around 45°, the missing coordination being instead provided by a chloride ion .
in any case, the zn-binding mode of hs100a <dig> is very similar to that of the four other s <dig> proteins for which crystal structures in the presence of zinc have been reported, namely s100a <dig>  s100a <dig>  s100a <dig>  and s100b  , with the particularity that s100a <dig> is the sole among these s <dig> proteins to display an all-histidine zinc binding site.
our structural data therefore clearly confirm that hs100a <dig> belongs to the his-zn group of s <dig> proteins.
an atypical zn-site formed by a 2-cacodylate complex
surprisingly, in the structure derived from crystal form  <dig>  four zinc ions per homodimer were present in addition to the four calcium ions .
a closer inspection of the structure revealed that only one of the two symmetrical zn-sites found in crystal form  <dig> was strictly conserved  whereas the second site  was instead replaced by a 2-cacodylate complex, the cacodylate molecule coming from the crystallization condition .
in this new site, zn <dig>  occupies almost the same position as zn <dig> from crystal form  <dig> and is coordinated by his <dig> from monomer a and his <dig> from monomer b. the two other coordinating his residues are however replaced by a chloride ion and by one of the hydroxyl groups of the cacodylate molecule.
instead, his <dig> from monomer a and his <dig> from monomer b are flipped by  <dig> and 100°, respectively.
as a consequence, his <dig> now coordinates the second zn2+  from the 2-cacodylate complex which is further held in place by his <dig> from monomer b, by a second chloride ion and by the second hydroxyl group of the cacodylate molecule.
finally, the new position of his <dig> leads to the creation of a third zn-site  which is completed by glu <dig> from the very c-terminal end of helix h <dig> , glu <dig> from a symmetry-related molecule, and a third chloride ion .
despite this quite large rearrangement of one of the zn-sites in crystal form  <dig>  the resulting s100a <dig> homodimer structure remains unaffected and superimposes well with the homodimer from crystal form  <dig>  with an overall root-mean-square deviation  on cα atoms of  <dig>  Å. hence it seems possible to accommodate more than just one zn2+ ion at the dimer interface within s100a <dig>  and, in addition to the four conserved histidines from the canonical zn-binding motif, other residues such as his <dig> or glu <dig> can participate in zinc coordination.
whether such additional zn2+ sites occur in vivo and what their functional relevance might be still remains to be determined.fig.
<dig> the atypical 2-cacodylate complex present in crystal form  <dig> induces the formation of new zn-sites.
a superimposition of the hs100a <dig> homodimers from crystal form  <dig>  and crystal form  <dig> , respectively.
only one zn-site  is conserved between the two structures.
b close-up view on the new zn-sites observed in the structure derived from crystal from  <dig>  the presence of the 2-cacodylate complex induces ligand swapping and the formation of two novel zn-sites
influence of divalent cations on s100a <dig> oligomerization
the zn2+/ca2+-hs100a <dig> homodimer we observe is highly similar to the previously reported ca2+-hs100a <dig> homodimer  <cit>  with an r.m.s.d.
on cα atoms of  <dig>  and  <dig>  Å between the ca2+-loaded homodimer and the zn2+/ca2+-bound homodimers from crystal forms  <dig> and  <dig>  respectively .
the most prominent changes are observed for the c-terminal part of helix h <dig>  which even forms an additional helix turn in the model derived from crystal form  <dig>  in crystal form  <dig>  helix h <dig> is not involved in crystal packing.
in crystal form  <dig>  helix h <dig> only interacts with glu <dig> from a symmetry-related molecule due to the presence of the 2-cacodylate complex.
the movement observed for helix h <dig> in both crystal forms is therefore more likely due to the presence of the zn sites rather than to a different crystal packing as compared to ca2+-hs100a <dig>  minor changes, mostly side chain repositioning of residues near the zinc binding pocket, are also noted around the two calcium ef-hands.
zinc binding was reported to increase both calcium affinity and target recognition for several s <dig> proteins, including s100b and s100a <dig>  <cit> .
based on structural data, it was proposed that increased target recognition is due to the repositioning of helix h <dig> c-terminus, which is believed to delineate the substrate binding cleft for many s <dig> proteins, whereas higher affinity for calcium is due to the stabilization of the ca2+-bound ef-hands active conformation  <cit> , although no major conformational changes are observed around the ef-hands between the ca2+-bound form and the zn2+/ca2+-bound form of both s100b and s100a <dig>  in our crystal structures of zn2+/ca2+-hs100a <dig>  the two calcium ef-hands superimpose well with the ef-hands of ca2+-hs100a <dig>  <cit> , as shown in fig.
3a, and no major side chain repositioning is observed except for the residues involved in zinc coordination.
however, a closer inspection of the ca-o distances between the calcium ions and their coordinating residues present in the ef-hands of both ca2+-hs100a <dig> and our two zn2+/ca2+-hs100a <dig> structures reveals that several of these ca-o distances are significantly shortened upon zn-binding .
such direct comparison is possible since all three structures were solved at similar resolutions around 2 Å. thus calcium binding by hs100a <dig> seems to be tighter in the presence of zinc.
these observations taken together with the fact that the changes observed in hs100a <dig> upon zinc binding are very similar to those described for s100b and s100a <dig> suggest that calcium affinity could be enhanced by zn2+ for hs100a <dig>  as we do not know where interacting partners would bind on hs100a <dig>  we cannot conclude whether zn2+ could also have an effect on target recognition for s100a <dig> or not.fig.
<dig> oligomeric states of hs100a <dig> in the presence of divalent cations.
a superimposition of the zn2+/ca2+-bound hs100a <dig> homodimers from crystal form  <dig>  and crystal form  <dig>  with ca2+-hs100a <dig>   <cit> .
the major regions of divergence are indicated with red arcs.
b elution profile of hs100a <dig> on a 24 ml superdex  <dig> size exclusion chromatography column  equilibrated in 20 mm hepes ph  <dig> , 200 mm nacl, 5 mm cacl <dig>  ca2+-hs100a <dig> elutes as two peaks with elution volumes of  <dig>  and  <dig>  ml, respectively, most likely corresponding to a tetramer and a dimer, respectively.
c superimposition of the zn2+/ca2+-bound hs100a <dig> homotetramers from crystal form  <dig>  and crystal form  <dig>  with the homotetramer derived from the crystal packing in ca2+-hs100a <dig>  and with the  <dig> heterotetramer .
the red arrow indicates the movement of one s100a <dig> homodimer away from the other homodimer on one side of the dimer-dimer interface in the calcium-loaded hs100a <dig>  <cit> .
d close-up view on the interactions stabilizing the homodimer-homodimer packing within the hs100a <dig> homotetramer from ca2+-hs100a <dig>  and from zn2+/ca2+-hs100a <dig>
the ca-o distances for which a significant change is observed upon zn2+ presence are highlighted in blue
oligomerization of s <dig> proteins strongly relies on divalent cations  <cit> .
as for many other s <dig> proteins, s100a <dig> and s100a <dig> can readily form homo- and heterodimers in the absence of calcium  <cit>  but the formation of the  <dig> heterotetramer is strictly ca2+-dependent  <cit> .
since the  <dig> heterotetramer is the most abundant form for the two proteins in vivo, most studies to evaluate the influence of divalent cations on s100a8/a <dig> have been performed on this oligomer.
thus little is known about the behaviour of s100a <dig> and/or s100a <dig> homo-oligomers in the presence of zinc, despite the fact that these forms have functions of their own  <cit>  that might also be regulated by divalent cations.
in the sole presence of calcium, we observe that hs100a <dig> elutes as two separate oligomeric forms on a size exclusion chromatography  column .
based on the calibration curve of the sec column , we calculated that these two forms respectively encompass  <dig>   and  <dig>   s100a <dig> homodimers, and we therefore assigned them as homotetramer and homodimer, respectively.
thus, s100a <dig> homodimers can further associate into homotetramers during sec.
this tetrameric form was already noticeable in the crystal packing of the ca2+-hs100a <dig> structure  <cit>  although the authors did not comment on it.
we observe a similar tetramer in our two distinct crystal forms  and the total buried surface area at the dimer-dimer interface is 1024 Å <dig> per dimer according to pisa  <cit> .
interestingly, the same tetrameric arrangement is observed for the  <dig> heterotetramer  <cit>  and all four tetramers superimpose quite well  suggesting that this tetramer is not dependent on very specific crystallization conditions and therefore may correspond to the tetramer observed in our size exclusion chromatography experiment.
within the hs100a <dig> homotetramer, the two homodimers are held in place by two symmetrical interfaces formed by the insertion of the second ef-hand from monomer b into the cleft between the two ef-hands of monomer a from the opposite homodimer .
in particular, the carbonyl groups of the main chain of lys <dig>  glu <dig>  asp <dig> and ile <dig>  interact with the side chains of either asn <dig> or tyr <dig>  while gln <dig> carbonyl group  connects with the side chain of asn <dig> .
interestingly, in ca2+-hs100a <dig>  the second hs100a <dig> homodimer is pulled away from the interface on one side, due to the slightly different orientation of helix h <dig> .
as a consequence, one of the two homodimer-homodimer interfaces is strongly destabilized due to the breaking of the hydrogen bonds made by asn <dig> side chain and the lengthening of all the other interactions described above .
these observations suggest that zinc further enhances stabilization of the s100a <dig> tetramer.
CONCLUSIONS
here we report the crystal structure of human s100a <dig> in the presence of both calcium and zinc.
the structure reveals an all-histidine zn-binding site and demonstrates that hs100a <dig> belongs to the his-zn group of s <dig> proteins, as expected from sequence comparisons.
the structure further reveals that zinc allows tightening of the dimer-dimer interface within hs100a <dig> homotetramer, thus pointing towards an enhancing role of zinc in s100a <dig> tetramerization.
our data therefore suggest that, as for several other s <dig> proteins, divalent cations, and notably zinc, influence s100a <dig> oligomerization.
the second zn2+/ca2+-hs100a <dig> structure presented here describes how an atypical 2-cacodylate complex can insert into one of the canonical zn-sites and disrupt the classical his <dig> tetracoordination sphere, inducing ligand swapping and the formation of two new zn-sites.
this is not unique to s100a <dig> as a ph-dependent ligand swapping for zinc coordination has also been described for s100b  <cit> .
furthermore, both s100a <dig> and s100a <dig> contains several histidine residues on their c-termini and they can all be involved in the coordination of divalent cations as seen in our s100a <dig> structures or in the structures of mn2+-bound calprotectin  <cit> .
taken together with our structural data, these observations suggest that the histidine-rich c-termini of both s100a <dig> and s100a <dig> are quite flexible and prone to ligand swapping in the presence of divalent cations.
reorganization of the canonical zn-sites leading to the involvement of new zn-coordinating residues and/or the creation of new zn-sites, possibly at the interface between two s <dig> dimers or between s100a <dig> and a binding partner, is a plausible scenario that could also occur in vivo under specific conditions.
ligand swapping in these flexible c-terminal regions could therefore potentially contribute to the formation of novel interdimer interactions, through ions coordination, that would lead to the formation of higher oligomeric species.
similarly, reorganization of the flexible c-terminus of both s100a <dig> and s100a <dig> by ligand swapping could induce the formation of cation binding sites at the interface between these s <dig> proteins and specific binding targets, the metal coordination being provided by both interacting partners.
such mechanisms would provide a structural rationale for the influence of zinc on s100:target recognition, as well as on the formation of s100a8/a <dig> amyloid-like structures which is known to be regulated by both calcium and zinc  <cit> .

 s proteins belong to the ef-hand calcium-binding protein superfamily and count more than members that are expressed exclusively in vertebrates, in a tissue- and cell-specific manner. under homeostatic conditions, they are found in the cytoplasm or in the nucleus of eukaryotic cells where they regulate vital processes, generally in a calcium-dependent manner. s protein expression is often upregulated during inflammation. in addition, these proteins can be secreted or passively released in the extracellular matrix where they become damage-associated molecular patterns and exert cytokine-like functions, through specific membrane-bound receptors, thus promoting sustained inflammation and tissue damage in various pathological settings associated with cardiovascular complications, neurodegenerative disorders and cancers. as a consequence, s proteins are considered both as valuable biological markers and important therapeutical targets for various inflammatory conditions. s100a and s100a constitute two of the most potent pro-inflammatory molecules among the s protein family. they are constitutively expressed by leukocytes of myeloid lineage. although products of distinct genes, the two proteins are often co-expressed and account for up to 45 % of the total cytosolic protein pool in human neutrophils. as for all their congeners, s100a and s100a form homodimers as a minimal, active unit. however, they usually exert their physiological function as a s100a8/a heterodimer, also termed calprotectin, and they can further associate via heterotetramerization. the s100a8/a heterodimer is proposed to regulate various homeostatic processes including fatty acid transport, cytoskeleton reorganization and myeloid cell differentiation. s100a and s100a have no signal sequences for classical secretion, but can be released from activated leukocytes, either by active secretion via a tubulin-dependent pathway, or by passive release, within neutrophil extracellular traps or from necrotic cells. as for all s proteins, s100a s100a and their heterodimer bind two calcium ions per monomer, one in each ef-hand motif. in addition to calcium, calprotectin can bind zn2+, mn2+, cu2+, and fe2+ and, by efficiently sequestrating these divalent cations in the extracellular environment, it acts as a potent antimicrobial agent. interestingly, this antibacterial property is restricted to the s100a8/a heterodimer although both homodimers are expected to bind zinc as well, based on sequence comparisons. indeed, zinc binding has been reported for several members of the s protein family and the determination of the three-dimensional structures of both s100b, s100a s100a and s100a complexed to zinc has revealed two common, symmetrical zinc binding sites at the dimer interface formed by highly conserved histidines and possibly one or two aspartate/glutamate. these zn2+-coordinating residues are conserved in s100a and s100a suggesting that both proteins could bind zinc in a similar fashion. however, no structural data are yet available to precisely characterize the zinc binding mode of either s100a and s100a homodimers or s100a8/a heterodimer. zinc was furthermore shown to enhance the oligomerization of these proteins, either as homo- and heterotetramers or as much more complex amyloid structures. finally, it has been proposed that the activity of extracellular s proteins, including s100a and s100a would be regulated by other divalent cations than calcium, notably zn2+, since the two ef-hand ca2+-binding sites would be fully saturated at all time in the extracellular compartment due to the high calcium concentrations. zinc appears therefore as an important regulator of the structural organization and the resulting functional properties of both s100a and s100a however, no structural information is yet available to decipher the intimate mechanisms by which this regulation is achieved. to better understand how zinc modulates the structure-function relationship of s100a and s100a we have undertaken the structural studies of these s proteins in the presence of zinc. here we report the crystallographic structures of human s100a bound to zinc and calcium at and  Å, respectively. the first structure reveals a classical, all-histidine zinc-binding motif, thereby classifying s100a as a genuine his-zn s protein. the second structure displays an atypical 2-cacodylate complex in one of the two zn-sites per homodimer, leading to ligand swapping and creation of two novel zn-sites. analysis of the quaternary arrangement of hs100a in both crystal forms suggests that zinc enhances tetramer stabilization and could further participate in the formation of higher oligomeric species and/or in the interaction with s100a binding partners.  a classical his-zn binding motif in the presence of both zinc and calcium, hs100a gave rise to two new crystal forms displaying a p and a c symmetry, as compared to ca2+-bound hs100a which crystallized in p . both structures could nevertheless be solved by molecular replacement using the previously reported ca2+-hs100a homodimer structure. for crystal form, structure determination was also successful using sad-phasing in phenix.autosolve from the dataset collected at the zn peak. in that case, sites were identified corresponding to the eight zn2+ ions and four of the eight ca2+ ions. the overall figures of merit after sad-phasing and after density modification using resolve were and, respectively. in any case, the position of the zinc ions could be unambiguously identified in both structures from anomalous difference fourier maps calculated using phases and figure of merit weights from the best refined atomic model containing no ions obtained with the native dataset and anomalous differences from the datasets collected at wavelengths of and  Å.table data collection and refinement statistics     i/σ   r meas values for the outer shell are given in parentheses fig. the crystallographic structure of zn2+/ca2+-bound hs100a a anomalous difference fourier maps calculated using phases and weight from the best refined atomic model obtained with the native dataset and anomalous differences from the datasets collected at wavelengths of  Å and  Å. the final model displayed as cartoon in beige and purple is superimposed for comparison. the anomalous signal clearly disappears for the wavelength above the zn maximal absorbance peak as judged by the fact that almost no density is visible for the second anomalous difference fourier map. b overview of the structure of zn2+/ca2+-bound hs100a derived from crystal form at  Å resolution showing two zinc bound at the homodimer interface. the four calcium ions are displayed as green spheres. c close-up view on the zn2+-binding site revealing an all-his binding motif formed by two residues from each monomer. d in two out of eight molecules within the asymmetric unit, his from monomer a is replaced by a chloride ion the overall structure of zn2+/ca2+-bound hs100a from crystal form is displayed in fig. 1b. the protein arranges in a canonical homodimer that contains four calcium ions, one in each ef-hand, and two zinc ions at the interface between the two monomers. although zinc can replace calcium in the ef-hand motifs when present alone, only ca2+ was found in all ef-hands of our model as judged from the anomalous difference fourier maps. the two symmetrical zn-sites are formed by two residues from each monomer: from the first monomer, his in helix h and his in the first ef-hand, and from the second monomer, his and his at the c-terminus of helix h. his appears however somewhat flexible and in two out of the eight molecules present in the asymmetric unit of our structure, it is flipped away from the zn-site by around 45°, the missing coordination being instead provided by a chloride ion. in any case, the zn-binding mode of hs100a is very similar to that of the four other s proteins for which crystal structures in the presence of zinc have been reported, namely s100a s100a s100a and s100b, with the particularity that s100a is the sole among these s proteins to display an all-histidine zinc binding site. our structural data therefore clearly confirm that hs100a belongs to the his-zn group of s proteins. an atypical zn-site formed by a 2-cacodylate complex surprisingly, in the structure derived from crystal form four zinc ions per homodimer were present in addition to the four calcium ions. a closer inspection of the structure revealed that only one of the two symmetrical zn-sites found in crystal form was strictly conserved whereas the second site was instead replaced by a 2-cacodylate complex, the cacodylate molecule coming from the crystallization condition. in this new site, zn occupies almost the same position as zn from crystal form and is coordinated by his from monomer a and his from monomer b. the two other coordinating his residues are however replaced by a chloride ion and by one of the hydroxyl groups of the cacodylate molecule. instead, his from monomer a and his from monomer b are flipped by and 100°, respectively. as a consequence, his now coordinates the second zn2+ from the 2-cacodylate complex which is further held in place by his from monomer b, by a second chloride ion and by the second hydroxyl group of the cacodylate molecule. finally, the new position of his leads to the creation of a third zn-site which is completed by glu from the very c-terminal end of helix h, glu from a symmetry-related molecule, and a third chloride ion. despite this quite large rearrangement of one of the zn-sites in crystal form the resulting s100a homodimer structure remains unaffected and superimposes well with the homodimer from crystal form with an overall root-mean-square deviation on cα atoms of  Å. hence it seems possible to accommodate more than just one zn2+ ion at the dimer interface within s100a and, in addition to the four conserved histidines from the canonical zn-binding motif, other residues such as his or glu can participate in zinc coordination. whether such additional zn2+ sites occur in vivo and what their functional relevance might be still remains to be determined.fig. the atypical 2-cacodylate complex present in crystal form induces the formation of new zn-sites. a superimposition of the hs100a homodimers from crystal form and crystal form, respectively. only one zn-site is conserved between the two structures. b close-up view on the new zn-sites observed in the structure derived from crystal from the presence of the 2-cacodylate complex induces ligand swapping and the formation of two novel zn-sites influence of divalent cations on s100a oligomerization the zn2+/ca2+-hs100a homodimer we observe is highly similar to the previously reported ca2+-hs100a homodimer with an r.m.s.d. on cα atoms of and  Å between the ca2+-loaded homodimer and the zn2+/ca2+-bound homodimers from crystal forms and respectively. the most prominent changes are observed for the c-terminal part of helix h which even forms an additional helix turn in the model derived from crystal form in crystal form helix h is not involved in crystal packing. in crystal form helix h only interacts with glu from a symmetry-related molecule due to the presence of the 2-cacodylate complex. the movement observed for helix h in both crystal forms is therefore more likely due to the presence of the zn sites rather than to a different crystal packing as compared to ca2+-hs100a minor changes, mostly side chain repositioning of residues near the zinc binding pocket, are also noted around the two calcium ef-hands. zinc binding was reported to increase both calcium affinity and target recognition for several s proteins, including s100b and s100a. based on structural data, it was proposed that increased target recognition is due to the repositioning of helix h c-terminus, which is believed to delineate the substrate binding cleft for many s proteins, whereas higher affinity for calcium is due to the stabilization of the ca2+-bound ef-hands active conformation, although no major conformational changes are observed around the ef-hands between the ca2+-bound form and the zn2+/ca2+-bound form of both s100b and s100a in our crystal structures of zn2+/ca2+-hs100a the two calcium ef-hands superimpose well with the ef-hands of ca2+-hs100a, as shown in fig. 3a, and no major side chain repositioning is observed except for the residues involved in zinc coordination. however, a closer inspection of the ca-o distances between the calcium ions and their coordinating residues present in the ef-hands of both ca2+-hs100a and our two zn2+/ca2+-hs100a structures reveals that several of these ca-o distances are significantly shortened upon zn-binding. such direct comparison is possible since all three structures were solved at similar resolutions around 2 Å. thus calcium binding by hs100a seems to be tighter in the presence of zinc. these observations taken together with the fact that the changes observed in hs100a upon zinc binding are very similar to those described for s100b and s100a suggest that calcium affinity could be enhanced by zn2+ for hs100a as we do not know where interacting partners would bind on hs100a we cannot conclude whether zn2+ could also have an effect on target recognition for s100a or not.fig. oligomeric states of hs100a in the presence of divalent cations. a superimposition of the zn2+/ca2+-bound hs100a homodimers from crystal form and crystal form with ca2+-hs100a. the major regions of divergence are indicated with red arcs. b elution profile of hs100a on a 24 ml superdex size exclusion chromatography column equilibrated in 20 mm hepes ph , 200 mm nacl, 5 mm cacl ca2+-hs100a elutes as two peaks with elution volumes of and  ml, respectively, most likely corresponding to a tetramer and a dimer, respectively. c superimposition of the zn2+/ca2+-bound hs100a homotetramers from crystal form and crystal form with the homotetramer derived from the crystal packing in ca2+-hs100a and with the heterotetramer. the red arrow indicates the movement of one s100a homodimer away from the other homodimer on one side of the dimer-dimer interface in the calcium-loaded hs100a. d close-up view on the interactions stabilizing the homodimer-homodimer packing within the hs100a homotetramer from ca2+-hs100a and from zn2+/ca2+-hs100a the ca-o distances for which a significant change is observed upon zn2+ presence are highlighted in blue oligomerization of s proteins strongly relies on divalent cations. as for many other s proteins, s100a and s100a can readily form homo- and heterodimers in the absence of calcium but the formation of the heterotetramer is strictly ca2+-dependent. since the heterotetramer is the most abundant form for the two proteins in vivo, most studies to evaluate the influence of divalent cations on s100a8/a have been performed on this oligomer. thus little is known about the behaviour of s100a and/or s100a homo-oligomers in the presence of zinc, despite the fact that these forms have functions of their own that might also be regulated by divalent cations. in the sole presence of calcium, we observe that hs100a elutes as two separate oligomeric forms on a size exclusion chromatography column. based on the calibration curve of the sec column, we calculated that these two forms respectively encompass and s100a homodimers, and we therefore assigned them as homotetramer and homodimer, respectively. thus, s100a homodimers can further associate into homotetramers during sec. this tetrameric form was already noticeable in the crystal packing of the ca2+-hs100a structure although the authors did not comment on it. we observe a similar tetramer in our two distinct crystal forms and the total buried surface area at the dimer-dimer interface is 1024 Å per dimer according to pisa. interestingly, the same tetrameric arrangement is observed for the heterotetramer and all four tetramers superimpose quite well suggesting that this tetramer is not dependent on very specific crystallization conditions and therefore may correspond to the tetramer observed in our size exclusion chromatography experiment. within the hs100a homotetramer, the two homodimers are held in place by two symmetrical interfaces formed by the insertion of the second ef-hand from monomer b into the cleft between the two ef-hands of monomer a from the opposite homodimer. in particular, the carbonyl groups of the main chain of lys glu asp and ile interact with the side chains of either asn or tyr while gln carbonyl group connects with the side chain of asn. interestingly, in ca2+-hs100a the second hs100a homodimer is pulled away from the interface on one side, due to the slightly different orientation of helix h. as a consequence, one of the two homodimer-homodimer interfaces is strongly destabilized due to the breaking of the hydrogen bonds made by asn side chain and the lengthening of all the other interactions described above. these observations suggest that zinc further enhances stabilization of the s100a tetramer.  here we report the crystal structure of human s100a in the presence of both calcium and zinc. the structure reveals an all-histidine zn-binding site and demonstrates that hs100a belongs to the his-zn group of s proteins, as expected from sequence comparisons. the structure further reveals that zinc allows tightening of the dimer-dimer interface within hs100a homotetramer, thus pointing towards an enhancing role of zinc in s100a tetramerization. our data therefore suggest that, as for several other s proteins, divalent cations, and notably zinc, influence s100a oligomerization. the second zn2+/ca2+-hs100a structure presented here describes how an atypical 2-cacodylate complex can insert into one of the canonical zn-sites and disrupt the classical his tetracoordination sphere, inducing ligand swapping and the formation of two new zn-sites. this is not unique to s100a as a ph-dependent ligand swapping for zinc coordination has also been described for s100b. furthermore, both s100a and s100a contains several histidine residues on their c-termini and they can all be involved in the coordination of divalent cations as seen in our s100a structures or in the structures of mn2+-bound calprotectin. taken together with our structural data, these observations suggest that the histidine-rich c-termini of both s100a and s100a are quite flexible and prone to ligand swapping in the presence of divalent cations. reorganization of the canonical zn-sites leading to the involvement of new zn-coordinating residues and/or the creation of new zn-sites, possibly at the interface between two s dimers or between s100a and a binding partner, is a plausible scenario that could also occur in vivo under specific conditions. ligand swapping in these flexible c-terminal regions could therefore potentially contribute to the formation of novel interdimer interactions, through ions coordination, that would lead to the formation of higher oligomeric species. similarly, reorganization of the flexible c-terminus of both s100a and s100a by ligand swapping could induce the formation of cation binding sites at the interface between these s proteins and specific binding targets, the metal coordination being provided by both interacting partners. such mechanisms would provide a structural rationale for the influence of zinc on s100:target recognition, as well as on the formation of s100a8/a amyloid-like structures which is known to be regulated by both calcium and zinc. 
here we report two crystallographic structures of calcium and zinc-loaded human s100a s100a binds two zinc ions per homodimer, through two symmetrical, all-his tetracoordination sites, revealing a classical his-zn binding mode for the protein. s proteinsef-handcalciumzincoligomerizationhttp://dx.doi.org/http://dx.doi.org/ /501100003554lundbeckfondenlundbeck foundation nanomedicine centre for individualized management of tissue damage and regenerationhttp://dx.doi.org/http://dx.doi.org/ /100008363kræftens bekæmpelsekortlægning på det atomare niveau af en pro-inflammatorisk og cancer fremmende signaleringsvej: krystalstrukturer af rage-ligand komplekserissue-copyright-statement© the author 2016 finally, we describe the calcium-induced s100a tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface. among these, s100a and s100a are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. furthermore, the presence of a 2-cacodylate complex in our second crystal form induces ligand swapping within the canonical his zinc binding motif, thereby creating two new zn-sites, one of which involves residues from symmetry-related molecules. despite this central role, no structural information on how zinc bind to s100a8/s100a and regulates their quaternary structure is yet available. furthermore, they show how zinc stabilizes s100a tetramerization and potentially mediates the formation of novel interdimer interactions. each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc.
 s proteins are a large family of calcium binding proteins present only in vertebrates. they function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. among these, s100a and s100a are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. in particular, zinc appears as a major regulator of s protein function in a disease context. despite this central role, no structural information on how zinc bind to s100a8/s100a and regulates their quaternary structure is yet available.  here we report two crystallographic structures of calcium and zinc-loaded human s100a s100a binds two zinc ions per homodimer, through two symmetrical, all-his tetracoordination sites, revealing a classical his-zn binding mode for the protein. furthermore, the presence of a 2-cacodylate complex in our second crystal form induces ligand swapping within the canonical his zinc binding motif, thereby creating two new zn-sites, one of which involves residues from symmetry-related molecules. finally, we describe the calcium-induced s100a tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.  our structures of zn2+/ca2+-bound hs100a demonstrate that s100a is a genuine his-zn s protein. furthermore, they show how zinc stabilizes s100a tetramerization and potentially mediates the formation of novel interdimer interactions. we propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords s proteinsef-handcalciumzincoligomerizationhttp://dx.doi.org/http://dx.doi.org/ /501100003554lundbeckfondenlundbeck foundation nanomedicine centre for individualized management of tissue damage and regenerationhttp://dx.doi.org/http://dx.doi.org/ /100008363kræftens bekæmpelsekortlægning på det atomare niveau af en pro-inflammatorisk og cancer fremmende signaleringsvej: krystalstrukturer af rage-ligand komplekserissue-copyright-statement© the author 2016 