S100A2 is an EF hand-containing Ca2+-binding protein of the family of S100proteins. The protein is localized exclusively in the nucleus and is involvedin cell cycle regulation. It attracted most interest by its function as a tumorsuppressor via p53 interaction. We determined the crystal structure ofhomodimeric S100A2 in the Ca2+-free state at 1.6-Å resolution. The struc-ture revealed structural differences between subunits A and B, especially inthe conformation of a loop that connects theN- andC-terminal EF hands andrepresents a part of the target-binding site in S100 proteins. Analysis of thehydrogen bonding network and molecular dynamics calculations indicatethat one of the two observed conformations is more stable. The structurerevealed Na+ bound to each N-terminal EF hand of both subunits coor-dinated by oxygen atoms of the backbone carbonyl and water molecules.Comparison with the structures of Ca2+-free S100A3 and S100A6 suggeststhat Na+ might occupy the S100-specific EF hand in the Ca2+-free state.
Keywords: S100A2; EF hand; X-ray structure; calcium; sodium
Introduction
With 21 members in humans, S100 proteins arethe largest subgroup within the family of EF handcalcium-binding proteins.1,2 They are small (Mr=10,000–12,000) acidic proteins containing an S100-specific N-terminal EF hand (14 residues) followedby a C-terminal classical EF hand (12 residues). Thetwo EF hands are connected by the so-called hingeloop. With the exception of S100G (calbindin D9k),all S100 proteins form homodimers and heterodi-mers under physiological conditions. Even highernoncovalent multimers are reported for S100A8/A9,S100A12, and S100B. Side chains of the C-terminalEF hand, including side chains of the hinge loop,represent the binding site for target proteins.3 Thesemostly acidic and hydrophobic residues comple-ment the basic and hydrophobic residues of thetarget molecule. The molecular interactions betweenS100 proteins and target peptides were elucidated bystructures of S100A10 in complex with a peptidederived from annexin II (1BT6), S100A11 in complexwith the N-terminus of annexin I (1QLS),4 S100Bbound to a peptide of p53 (1DT7),5 NDR kinase(1PSB),6 andTRTK-12 (1MWN).7 Detailed inspectionof the complexes shows that each of themhas distinctstructural characteristics concerning orientation and
ess:
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arrangement of the target in the binding pocket. Thisis rather unexpected, because the locations of severalhydrophobic residues in helices III and IVas well asnegatively charged residues in the hinge loop arewell conserved among S100 proteins.6 These struc-tures revealed the functional importance of the hingeloop, together with distinct residues in the helices ofthe C-terminal EF hand.S100 proteins show pronounced cell- and tissue-
specific expression patterns and are involved in dif-ferent physiological processes, such as cell cycleregulation, cell growth, differentiation, and motility.Consequentially, dysregulation of S100 protein ex-pression is associated with several diseases, espe-cially neurodegenerative and cardiovascular dis-orders, such as cancer.8 S100A2 is unique amongthe family of S100 proteins due to its predominantlynuclear localization.9 S100A2 shows a broad expres-sion pattern, including several tissues such as lung,kidney, liver, cardiac muscle, and skeletal muscle.10S100A2 was first identified as a tumor suppressor inhumanmammary epithelial cells.11 Reduced expres-sion levels are also observed in prostate adeno-carcinoma, lung cancer, and breast carcinoma.12–14
Recently, it was shown that S100A2 binds and activ-ates p53 in a Ca2+-dependent manner that directlylinks S100A2 and p53 tumor-suppressing activ-ities.15 Besides Ca2+, S100A2 also binds Zn2+ withhigh affinity (Kd=25 nM). In a recent study, weshowed that Zn2+ binding to S100A2 abolishes itsCa2+ affinity and thereby inhibits response to intra-
Fig. 1. Ribbon representation of the S100A2 dimer.Subunits A and B are shown in red and blue, respectively.The N-termini are shown in dark colors, whereas theC-termini are shown in bright colors. The Na+ ions aredepicted as green spheres.
934 Structure of Ca2+-Free S100A2
cellular Ca2+ signals.16 This implies that S100A2 isregulated by both Ca and Zn2+.To date, there is no three-dimensional structure of
S100A2 available. Multidimensional NMR studiesrevealed a molecular architecture similar to that ofother S100 proteins.17 Additionally, it was shownthat S100A2 exists in equilibrium of at least twoisoforms, most probably due to cis–trans isomerismof three proline residues that are located in the linkerregion and in the C-terminus, which hamperedthe determination of the structure by NMR.5 Up tonow, X-ray structures of Ca2+-free S100 proteinsare only available for S100A3 (1KSO) and S100A6(1K9P).18,19 Additionally, several structures of Ca-free S100 proteins have been determined by NMRspectroscopy: S100A1 (1K2H),20 S100A4 (1M31),21
S100A6 (2CNP),22 S100A11 (1NSH),23 S100A13(1YUS),24 S100B (1B4C),25 S100P (1OZO),26 andS100G (1CLB).27 The majority of structures of S100proteins represent the Ca2+-loaded state, which isthe active form of S100 proteins.28 It is essential toobtain structural information on both the Ca2+-freeand Ca2+-loaded states in order to identify and under-stand the structural rearrangements induced by Ca2+
binding. In this work, we present the structure ofS100A2 determined by X-ray crystallography in itsCa2+-free form at 1.6-Å resolution, providing de-tailed insights into the inactive form of an S100 protein.
Crystallization of S100A2
As reported in a previous study, the cysteine thiolsof S100A2wt are prone to oxidation forming intra-molecular and intermolecular disulfides. Due to thisoxidation, several attempts in crystallizing S100A2wtwere unsuccessful. Therefore, we chose the cysteine-deficient S100A2 variant (C2S–C21S–C86S–C93S) forcrystallization.29 Size-exclusion experiments exhib-ited that the protein elutes as one major peak corres-ponding to an S100A2 homodimer (22 kDa). Clearly,the cysteine-to-serine exchanges caused no majorchange concerning dimerization. CD analysis re-vealed that the secondary structure content of thevariant is virtually identical with that of S100A2wt,29
showing that the mutation of the four Cys residuesdid not affect the overall structure. Furthermore, theCa2 +-binding properties of several Cys-to-Ser var-iants were not affected compared with the wild-typeprotein,16 demonstrating that the functionality of theprotein is not changed.
Overall structure of S100A2
The final model comprises two subunits (A and B)containing residuesMetA1 to SerA93, residues SerB2to SerB93, and 171 water molecules. After severalsteps of refinement, the electron density differencemaps revealed the presence of three Na ions and oneisopropanol molecule. The last four C-terminal resi-dues (Pro94–Pro97) of both subunits could not beresolved. This might be due to structural hetero-geneity caused by cis–trans isomerization of theproline residues as indicated by NMR studies.30
Like other S100 proteins, one S100A2 subunit con-tains anN-terminal S100-specific EF hand flanked byhelix I (amino acids 4–20 in subunit A and aminoacids 5–20 in subunit B) and helix II (amino acids31–41) and a C-terminal classical EF hand betweenhelix III (amino acids 52–64 in subunit A and aminoacids 53–64 in subunit B) and helix IV (amino acids72–86), which are linked by the so-called hinge re-gion (Fig. 1).28
Additionally, the C-terminus contains a short α-helical segment comprising residues 88–91, whereasthe corresponding residues in the structures ofCa2+-free S100A3 and Ca2+-free S100A6 form a 310-helix. Structure validation of Ca2+-free S100A2 ex-hibits that 95.2% of the residues reside in the mostfavored regions and that 4.8% reside in additionalallowed regions. The surface representation of thehomodimer shows an arc-shapedmolecule. The con-cave molecule surface is dominated by a negativelycharged electrostatic surface, whereas the convexsurface exhibits some additional positively chargedpatches (Fig. 2). Other Ca2+-free S100 proteins alsoexhibit a similar shape, but the surface charge distri-bution is quite different. S100A1 (1K2H) and rat S100B(1B4C) are dominated by a negatively charged sur-face. However, S100A4 (1M31) is dominated by apositively charged surface, despite the high aminoacid sequence identity to S100A2 (61.5%). S100A3(1KSO) is characterized by equally distributedcharges on the surface.
Comparison of Ca2+-free S100A2 with Ca2+-freeS100A3, S100A4, and S100A6.
The structure of Ca2+-free S100A2 shows remark-able similarity to the X-ray structures of Ca2+-freeS100A318 (Cα 4–41, 52–92; r.m.s.d.=0.69 Å) and Ca2+-
Fig. 2. Electrostatic surface representation of the S100A2 dimer. The electrostatic surface potential was calculated byAPBS.31 Red and blue areas indicate negatively (−9.0 kT/e) and positively (+9.0 kT/e) charged regions, respectively.Three views of the molecule are shown rotated in increments of 90°.
935Structure of Ca2+-Free S100A2
free S100A6 (Cα 4–41, 52–90; r.m.s.d.=0.64 Å),19 aswell as toCa2+-free S100A4 (Cα 4–41, 52–91; r.m.s.d.=2.33 Å), determined by NMR spectroscopy (Figs. 3band c).21 The conformations of theCa2+-free EF handsare very similar as illustrated by the interhelicalangles in each of the EF hands (Table 1). In addition,the overall topology is well conserved as shown bythe overall structural superposition.Major differencesoccur in the hinge regions displaying r.m.s.d. valuesof approximately 4 Å (Fig. 3).Vallely et al.21 proposed that S100A2, S100A3,
S100A4, and S100A6 may adopt similar helix III/helix IV interhelical angles in the apo state. Theirproposal was based on the observation that in apo-S100A4, Ala54 (helix III) and Met85 (helix IV) form ahydrophobic contact stabilizing a particular inter-helical angle.21 These residues are conserved inS100A2, S100A3, and S100A6 but not in other S100proteins. We found that the corresponding residuesGly54 and Met85 indeed form a hydrophobiccontact in apo-S100A2 that is further stabilized byLeu55. The interhelical angles of the four S100 pro-teins are very similar as illustrated in Fig. 3b. Allinterhelical angles and distances are given in Table 1.
Comparison of the two subunits
In several X-ray structures of S100 proteins, suchas bovine S100B [Protein Data Bank (PDB) code1MHO],32 human S100A6 (PDB codes 1K96 and1K8U),19 and S100A7 (PDB code 2PSR),33 the 2-foldsymmetry axis in the S100 protein dimer representsa crystallographic axis (i.e., both subunits adopt anidentical conformation in the crystal). The asym-metric unit of the apo-S100A2 crystal contains bothsubunits. As expected, both subunits are overallquite similar although superposition of subunit A onsubunit B gives an overall r.m.s.d. of 2.73 Å (Cα 4–92). If the hinge region is excluded (Cα 4–41, 52–92),the similarity of the two subunits is more obviouswith an r.m.s.d. of 0.37 Å. The conformation of thehinge region in both subunits is noticeably different,yielding r.m.s.d. values for the hinge loop Cαpositions (residues 42–52) between 0.42 and 7.05 Å.These deviations are illustrated in a superposition ofthe two subunits (Fig. 3a). Furthermore, smallchanges in the secondary structure of both subunits
were observed. In subunit A, helix III comprisesresidues 52–64, whereas in subunit B, helix III isshorter by one residue (amino acids 53–64) due to amissing hydrogen bond between the carbonyl groupof AspB51 and the amide group of LeuB55; again, insubunit B, helix I (amino acids 5–20) is shorter byone residue compared with that in subunit A (aminoacids 4–20). The differences between the subunitsare also illustrated by deviations in the interhelicalangles of both subunits (summarized in Table 1),which account for aΔ of 1.8° between the helix I andhelix II pairs and aΔ of 2.6° between the helix III andhelix IV pairs. The largest deviation with a Δ of 5.4°is observed for the helix II/helix III pairs. Theindividual interhelical angles and midpoint dis-tances of the helices of both subunits are given inTable 1.
Comparison of the hinge regions of bothsubunits
It has been shown that residues of the hinge regionand the C-terminus of S100 proteins form the targetinteraction site.4,5,7,34–36 The hinge regions in bothsubunits of Ca-free S100A2 adopt two completelydifferent conformations. The available NMR struc-tures of apo-S100 proteins20–22,25 revealed somedegree of conformational flexibility in the hingeregion. However, the analysis is complicated by theoverlap of the signals originating from both subunitsthat results in signal broadening (W.J. Chazin,personal communication, 2007, G. Fritz, Ed.; D.J.Weber, personal communication, 2007, G. Fritz, Ed.).We therefore analyzed the structure of the hingeregions in order to get insights on whether there is afavored conformation. Figure 3a illustrates theobvious differences in the conformations of thehinge regions. In subunit A, it exhibits a pronouncedextension forming a bend that points out of themolecule, whereas in subunit B, the loop points tothe inside of the molecule. Analysis of the average B-factors of residues Glu41–Glu51 gave 16.6 Å2 forsubunit A but an almost twofold higher value of28.7 Å2 for subunit B. Pronounced differences alsooccur in the hydrogen bonding pattern within thehinge regions (Glu41 to Asp51). The loop in subunitA is stabilized by six hydrogen bonds with residues
Fig. 3. (a) Superposition of the subunits of S100A2. (b) Structural alignment of S100A2 with S100A3 (1KSO), S100A4(1M31), and S100A6 (1K9P). Subunit A of S100A2 is shown in red; subunit B, in orange; S100A3, in green; and S100A6, inblue. (c) Amino acid sequence alignment of S100A2, S100A3, S100A4, and S100A6. The secondary structure elements werecalculated by DSSP and are indicated by colored rectangles: red=α-helix; magenta=310-helix; yellow=β-sheet. Residuesthat were not resolved in the structures are indicated in lowercase.
936 Structure of Ca2+-Free S100A2
in subunit A itself (e.g., between the amide nitrogenof GlyA47 and the carboxyl group of GluA52). Fur-ther stabilization of the loop might also be a result ofcrystal packing. There is one crystal contact of sub-unit A with a neighboring molecule in the crystal,comprising the amino group of LysA49 and the car-boxyl group of AspB88′ from the adjacent molecule(Fig. 4; Table 2). In subunit B, there are threehydrogen bonds to subunit B itself—two to subunitA and one due to a crystal contact between thecarboxyl group of AspB51 and the amide nitrogen of
AspA25′ of the neighboring molecule (Table 2).Additionally, to these direct protein–protein inter-actions, there is a vast network of hydrogen bonds tosubunit A itself as well as to other molecules in thecrystal by intermediate water molecules. Betweenresidues GluA41 and AspA51, there are 26 stabiliz-ing interactions by intermediate water molecules.However, only 7 stabilizing interactions by inter-mediate water molecules were observed in the cor-responding region of subunit B. It has been shownthat such water molecules acting as hydrogen bond
Table 1. Interhelical angles and midpoint distances in apo-S100A2 in comparison with S100A3, S100A4, and S100A6
a In the case of S100A6, there is only one subunit in the asymmetric unit.
937Structure of Ca2+-Free S100A2
bridges are important for the stabilization ofprotein–protein, protein–DNA, and protein–smallmolecule interactions.37–39
Furthermore, a Na+ ion bound in the hinge regionof subunit A might contribute to the stabilization ofthe observed conformation. The Na+ ion is coordi-nated by the carbonyl oxygen atoms of LeuA42 andHisA39, one carboxyl oxygen atom from GluA74′of an adjacent molecule, and one water molecule(Fig. 4).Thus, the conformation of the hinge region in
subunit A of apo-S100A2 is stabilized by hydrogenbonds and, to some part, by crystal contacts,whereas the hinge region of subunit B might adoptdifferent conformations within the crystal as indi-cated by the higher B-factors in this region.
Fig. 4. Stabilization of the hinge region in subunit A.The hinge region in subunit A (yellow) is stabilized by ahydrogen bond network. The majority of hydrogen bondsare formed between residues in subunit A itself. One saltbridge to DA88′ (green) of a neighboring molecule in thecrystals might also contribute to the stability of the loop inthe crystal structure. Additionally, a Na+ (magenta) iscoordinated by three residues of the hinge region bridgingsubunit A and one of the neighboring molecules in thecrystal (cyan).
Molecular dynamics analysis of Ca2+-freeS100A2
As outlined above, the conformation of the hingeregion in subunit A might be more stable than thatin subunit B. However, the interpretation of thestructural data is not straightforward because thepacking of the molecules in the crystal might alsocontribute to the stabilization of the hinge region insubunit A. In order to check our hypothesis that theconformation of the hinge region in subunit A ismore stable than that in subunit B, we performed amolecular dynamics analysis of the fully hydratedprotein. Water molecules identified in the X-raystructure were kept, and ca 12,000 water moleculeswere added. The sodium ion located close to thehinge region of subunit Awas removed. The r.m.s.d.values and dihedral angles of the main chain Cαatoms of residues 46–51 from both subunits wereextracted from the simulation data and are plottedin Fig. 5. During the first 700 ps of the simulation,there is no obvious difference in the flexibility ofboth hinge regions. However, after that period, thereare large variations in the r.m.s.d. and in thedihedral angle of the main chain in the hinge regionof subunit B (Fig. 5), whereas the hinge region ofsubunit A shows far smaller variations. Thus, thesimulation data support that the hinge region insubunit A adopts a more stable conformation thanthat in subunit B.The structural data show that conformational
variability is realized in S100A2 hinge loops. UponCa2+ binding, the S100 proteins undergo a majorconformational change, rotating helix HIII byapproximately 90° and repositioning the residuesin the hinge region. The observed variability in thehinge region might therefore significantly contribute
Table 2. Protein–protein H bonds formed by residuesGlu41–Asp51 in subunits A and B
Fig. 5. Protein backbone movements in the hingeregions of subunits A and B during molecular dynamicssimulation. (a) The average r.m.s.d. values of the Cα atoms(residues 46–51) from their initial positions are plottedversus time. The r.m.s.d. values were calculated for each 1-ps interval of the simulation. (b) The average dihedralangles of Cα atoms of residues 46–51 were calculated foreach 1-ps interval of the simulation. The r.m.s.d. (a) anddihedral angle (b) values for subunit A are depicted asblack squares, whereas those for subunit B are depicted asred circles. Both parameters for the hinge region in subunitA (black) show a time course with small amplitudesalmost parallel with the x-axis. In contrast, both r.m.s.d.and dihedral angle values for subunit B (red) exhibit acurved time course, documenting conformationalchanges.
Tabl
e3.
Na+–o
xyge
ndistan
cesin
S100
A2,
S100
A3,
andS1
00A6,
aswella
sthreefurthe
rproteins
forcompa
rison
S100A2A
d(Å)
O–N
a+S1
00A2B
d(Å)
O–N
a+S1
00A3
1KSO
d(Å)
O–O
S100A6
1K9P
d(Å)
O–O
1JZ8
d(Å)
O–N
a+1G
EN
d(Å)
O–N
a+1H
XN
d(Å)
O–N
a+
SerA
20–O
2.52
SerB20–O
2.46
AlaA20–O
3.13
Ser20–O
2.25
PheC
556–
O2.25
Ile478–O
2.39
Met23
8–O
2.23
GluA23–O
2.23
GluB2
3–O
2.34
CysA23
–O2.84
Glu23–O
2.47
TyrC
559–O
2.35
Val52
3–O
2.01
Ala28
3–O
2.27
Asp
A25
–O2.42
Asp
B25–
O2.49
Asp
A25–O
2.96
Asp
25–O
2.39
ProC
650–O
3.12
Ala57
1–O
2.13
Ala33
5–O
2.32
LysA
28–O
2.29
LysB
28–O
2.28
LysA
28–O
2.76
Thr28–O
2.41
LeuC
562–O
2.29
Val62–O
2.34
Ala37
8–O
2.39
WatA105
2.33
WatB1
072.52
Wat19
2.68
Wat26
2.58
Wat4410
2.48
Wat317
2.59
Phosph
ate41
2.85
WatA10
62.35
WatB1
092.29
Wat139
2.83
Wat47
2.68
Wat46
522.26
Chloride30
32.77
Chloride3
2.84
938 Structure of Ca2+-Free S100A2
to the energetics of the Ca2+-induced structuralchanges. So far, conformational variability of thehinge region in S100 proteins was deduced fromhigher average B-factors in X-ray structures or theabsence of nuclear Overhauser enhancements instructures determined by NMR. Only one study, byInman et al.,40 on apo-S100B showed that the hingeregion displays some degree of flexibility. Thestructure presented here shows for the first timedistinct conformations of the hinge region, whichmight display different levels of stability as indi-cated by the molecular dynamics study. Stabiliza-tion or destabilization of the inactive apo formdirectly affects the Ca2+ affinity of the protein, whichis the most important parameter for the activity ofthe protein. Thus, the evaluation of structuralfeatures contributing to the stability of EF handproteins can give important insights into the controlof Ca2+ affinity and physiological action.
Na+ bound in the N-terminal EF hands
In the structure of Ca2+-free S100A2, we observedthree metal ions. Each N-terminal S100-specific EFhand of both subunits bound one ion, and a further
939Structure of Ca2+-Free S100A2
ion was coordinated by residues to the hinge loop ofsubunit A. According to the Cambridge StructuralDatabase,41 sodium–ligand interactions in smallmolecule crystals are predominantly (∼90%) withoxygen atoms. Harding42,43 evaluated these smallmolecules from the Cambridge Structural Databaseand metal sites in protein structures in the PDB.These studies revealed a mean of 2.38 Å for sodium–main chain carbonyl distances and that of 2.41 Å forsodium–water distances.42,43 The mean distancefrom the metal ions to carbonyl oxygen is 2.38±0.11 Å and that from metal ions to water oxygen is2.37±0.10 Å, thus in good agreement with Na+
bound to the S100A2. The angles and bond lengthsfor the coordination of the Na+ ions are summarizedin Table 3. Although Mg2+ has the same number ofelectrons as Na+, it can be distinguished from Na+
by significantly shorter mean distances of 2.26 Å tocarbonyl oxygen and 2.01 Å to water oxygen, whichare not in agreement with the distances observed inS100A2. Furthermore, the crystallization buffercontained 100 mM Na+, whereas divalent cationshave been removed prior to crystallization.29
In the S100-specific EF hands, the Na+ is coordi-nated by the carbonyl oxygen atoms from Ser20,Glu23, Asp25, and Lys28. Additionally, two watermolecules complete the octahedral coordination(Fig. 6). The coordination resembles the bindingmode Ca2+ in the structures of Ca2+-loaded S100proteins. However, the glutamate (Glu33), which is akey residue in Ca2+ binding, is in a distance of about5 Å to the Na+ and not involved in coordination.Subunits A and B of S100A2 show slight differ-
ences between the sodium donor atom distances.The Na+–ligand distances of three further Na+ siteswith similar coordination are listed in Table 3 inorder to compare the Na+ coordination in S100A2with other proteins. In each protein, Na+ is coor-
Fig. 6. Representation of the Na+-binding site in theN-terminal EF hand of subunit A. An overlay of the finalFo−Fc electron density for the sodium ion (green) is shownin yellow with a sigma contour of 4.0.
dinated octahedrally by four backbone carbonyloxygen atoms and two additional water moleculesor negatively charged ions: in β-galactosidase (PDBcode 1JZ8), two water molecules are involved incoordination,44 whereas in human gelatinase A(PDB code 1GEN), there are one water moleculeand one chloride ion;45 in hemopexin (PDB code1HXN), a chloride ion and a phosphate ion completethe octahedral coordination of the bound Na+.46 Thedata in Table 3 illustrate that the sodium–oxygendistances can vary between 2.01 and 3.13 Å.Furthermore, the donor distances for charged ionsare increased compared with oxygen donor atoms.The X-ray structure of Ca2+-free S100A2 shows for
the first time coordination of Na+ in an EF hand. Ithas been reported earlier47,48 that Na can competewith Ca2+ for binding to EF hands, and a Kd of1.5 mM was determined for Na+ binding to the CDsite of rat apo-α-parvalbumin.48 The Na+ concentra-tion of approximately 50 mM in the crystallizationbuffer indicates that the Kd might be at least∼20 mM, because both Na+ ions in the N-terminalEF hands are fully occupied. In contrast to the N-terminal EF hand, no Na+ binding was observed tothe C-terminal EF hand. This might be readilyexplained by the different charge densities of thetwo EF hands. In the N-terminal S100-specific EFhand, only one glutamate side chain is involved inCa2+ coordination, whereas in the C-terminal classi-cal EF hand, the side chains of two aspartates, oneglutamate, and one asparagine coordinate the Ca2+.In the Ca2+-free state, the strong electrostaticrepulsion of the negatively charged side chainsinduces an extended conformation of the C-terminalCa2+-binding loop with the side chains pointing indifferent directions to the solvent. NMR analysis alsoindicates that the C-terminal EF hand ismore flexiblethan the N-terminal S100-specific EF hand (W.J.Chazin, personal communication, 2007, G. Fritz,Ed.).40 Because the side chains of the acidic residueshave to face each other in order to chelate an ion,effective compensation of the negative charges isachieved with divalent, but not monovalent, ions.Na coordination in the S100-specific EF hand is
most likely a consequence of the preformation of thisEF hand. All backbone carbonyls are already inplace for ion binding, and only minor structuralchanges are required in this site compared with theC-terminal classical site to bind Ca2+. This wasdemonstrated in great detail for S100G (calbindinD9k), where no substantial change in the localbackbone conformations in the apo form as well asCd2+- and Ca2+-loaded states was observed.49,50
It is tempting to ask whether the S100-specific EFhand is loaded with Na in the inactive Ca2+-freeform. So far, it was assumed that Mg2+ occupies theCa2+-binding sites of EF hand proteins when the cellis in the resting state, characterized by low intra-cellular Ca2+ concentrations. As soon as Ca2+
concentrations in the cell increase, the bound Mg2+
is replaced by Ca2+.51 The change from sixfold coor-dination of Mg to sevenfold coordination of Ca2+
induces structural changes and activation of the EF
940 Structure of Ca2+-Free S100A2
hand proteins. However, the S100-specific EF handdisplays some differences from the classical EF hand:it coordinates Ca2+ mainly via backbone carbonyloxygen and only one glutamate side chain.28 Notably,X-ray structural analysis of Mg2+ or Mn2+-loadedS100G revealed an Mg2+/Mn2+ ion in the C-terminalEF hand but not in the N-terminal EF hand.52 Theauthors also determined the Mg-binding affinity andshowed that S100G binds only one Mg2+ underphysiological conditions. The affinity of the S100-specific EF hand for Mg2+ in S100G was shown to bevery low and not occupied even at Mg2+ concentra-tions of 120 mM in the crystallization buffer. Exam-ination of the X-ray structures of Ca2+-free S100A3,S100A6, and S100G for potential Na+-binding sitesshowed that the N-terminal S100-specific EF handsharbor a well-ordered water molecule in a similar po-sition as the Na+ ion in S100A2. Analysis of the watermolecules by valence calculations using the programWASP53 revealed Na valences of 0.64 for the site inS100A3, 1.04 in S100A6, and 0.55 in S100G. The dataindicate that all three proteins might harbor a Na+ inthe N-terminal EF hand too. S100A6 displayed thehighest Na+; interestingly, the crystallization buffercontained 15–30 mM Na+,19 whereas the buffers forcrystallization of S100A318 and S100G52 containedonly traces of Na+.
Materials and Methods
Cloning, expression, and purification
The S100A2 variant S100A2–C2S–C21S–C86S–C93Swas obtained by site-directed mutagenesis and subse-quently cloned into the bacterial expression vectorpMW172 as described elsewhere.54 Expression in Escher-ichia coli BL21(DE3) and purification were carried out asdescribed previously.31 Briefly, expression was carried outin 50 mM phosphate-buffered DYT medium, pH 7.4, at310 K. Isolation of S100A2ΔCys was performed by two-step purification, including hydrophobic interaction chro-matography on a phenyl Sepharose Fast Flow column(100 ml, GE Healthcare). The second step was size-
Table 4. Data collection and refinement statistics
a The numbers in parentheses are the statistics for the highest-resolution shell.
b Rmerged–F (%) =∑(|AIh ,P−AIh ,Q|)/(0.5 *∑AIh ,P +AIh ,Q),according to Diederichs and Karplus.55
exclusion chromatography on a Superdex 75 (26/60)column (GE Healthcare).
Data collection
Data collection was carried out at Swiss Light Source(Paul Scherrer Insitute, Villigen, Switzerland) at beamlineX06SA PX using a mar225 mosaic CCD detector (MarResearch) (data statistics are summarized in Table 4).
Structure determination and refinement
Crystallographic data were processed using XDS.56
Crystals belonged to space group P212121, with unit celldimensions a=43.5 Å, b=57.8 Å, and c=59.8 Å containingtwo subunits per asymmetric unit. The structure wasdetermined by molecular replacement using the programPhaser version 1.3.1.57,58 The crystal structure of Ca2+-freeS100A3 (PDB code 1KSO)18 served as a search model. Therefinement of the model was carried out with data to 1.6-Åresolution using Refmac5.59 Iterative cycles of refinementand manual model building were performed with theprograms O60 and Coot61 until no improvement in thecrystallographic free R-factor was observed. Data collec-tion and refinement statistics are summarized in Table 4.Secondary structure content was assigned using DSSP.62
Structure validation was performed using SFCHECK63
and PROCHECK.64 Electrostatic potential calculationswere performed with the program APBS (AdaptivePoisson–Boltzmann Solver).31 Interhelical angles werecalculated using the program interhlx.65
Molecular dynamics
Molecular dynamics calculations of Ca-free S100A2were performed using GROMACS.66 A solvated box withedges 9 Å from the protein periphery contained ca 12,200water molecules and 14 sodium ions to neutralize the netcharge of the protein. Water molecules identified in thecrystal structure and the sodium ions in the N-terminal EFhand of both subunits were included. The third sodiumion bound at the hinge region of subunit Awas moved tothe periphery of the molecule. Energy minimization usingsteepest descent with GROMOS96 43a1 force field wasperformed until convergence b800 kJ mol nm−1 wasreached. Position-restrained molecular dynamics wasperformed for 20 ps to further equilibrate the solvent.Molecular dynamics simulation at 300 K was performedfor 2000 ps, and the results of the constant temperaturesimulation were visualized using VMD. The four Serresidues in each subunit, which had replaced the Cysresidues for crystallization, were mutated in silico back toCys, and the molecular dynamics runs were repeated. Nonoticeable effect of the Cys→Ser mutation was observed.
Accession number
Coordinates and structure factors have been depositedin the PDB with accession number 2RGI.
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.03.019
941Structure of Ca2+-Free S100A2
References1. Marenholz, I., Heizmann, C. W. & Fritz, G. (2004).
S100 proteins in mouse and man: from evolution tofunction and pathology (including an update of thenomenclature). Biochem. Biophys. Res. Commun. 322,1111–1122.
2. Marenholz, I., Lovering, R. C. & Heizmann, C. W.(2006). An update of the S100 nomenclature. Biochim.Biophys. Acta, 1763, 1282–1283.
3. Bhattacharya, S., Bunick, C. G. & Chazin, W. J. (2004).Target selectivity in EF-hand calcium binding pro-teins. Biochim. Biophys. Acta, 1742, 69–79.
4. Rety, S., Osterloh, D., Arie, J. P., Tabaries, S., Seeman, J.,Russo-Marie, F. et al. (2000). Structural basis of theCa2+-dependent association between S100C (S100A11)and its target, the N-terminal part of annexin I.Structure, 8, 175–184.
5. Rustandi, R. R., Baldisseri, D. M. &Weber, D. J. (2000).Structure of the negative regulatory domain of p53bound to S100B(betabeta). Nat. Struct. Biol. 7, 570–574.
6. Bhattacharya, S., Large, E., Heizmann, C. W., Hem-mings, B. & Chazin, W. J. (2003). Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100target specificity and activation of the kinase. Biochem-istry, 42, 14416–14426.
7. Inman, K. G., Yang, R., Rustandi, R. R., Miller, K. E.,Baldisseri, D. M. & Weber, D. J. (2002). Solution NMRstructure of S100B bound to the high-affinity targetpeptide TRTK-12. J. Mol. Biol. 324, 1003–1014.
8. Heizmann, C. W., Fritz, G. & Schäfer, B. W. (2002).S100 proteins: structure, functions and pathology.Front. Biosci. 7, d1356–d1368.
9. Mandinova, A., Atar, D., Schäfer, B. W., Spiess, M.,Aebi, U. & Heizmann, C. W. (1998). Distinct sub-cellular localization of calcium binding S100 proteinsin human smooth muscle cells and their relocation inresponse to rises in intracellular calcium. J. Cell Sci.111, 2043–2054.
10. Glenney, J. R., Jr., Kindy, M. S. & Zokas, L. (1989).Isolation of a new member of the S100 protein family:amino acid sequence, tissue, and subcellular distribu-tion. J. Cell Biol. 108, 569–578.
11. Lee, S. W., Tomasetto, C. & Sager, R. (1991). Positiveselection of candidate tumor-suppressor genes bysubtractive hybridization. Proc. Natl. Acad. Sci. USA,88, 2825–2829.
12. Gupta, S., Hussain, T., MacLennan, G. T., Fu, P., Patel,J. & Mukhtar, H. (2003). Differential expression ofS100A2 and S100A4 during progression of humanprostate adenocarcinoma. J. Clin. Oncol. 21, 106–112.
13. Feng, G., Xu, X., Youssef, E. M. & Lotan, R. (2001).Diminished expression of S100A2, a putative tumorsuppressor, at early stage of human lung carcinogen-esis. Cancer Res. 61, 7999–8004.
14. Wicki, R., Franz, C., Scholl, F. A., Heizmann, C. W. &Schäfer, B. W. (1997). Repression of the candidatetumor suppressor gene S100A2 in breast cancer ismediated by site-specific hypermethylation. CellCalcium, 22, 243–254.
15. Mueller, A., Schäfer, B. W., Ferrari, S., Weibel, M.,Makek, M., Hochli, M. & Heizmann, C. W. (2005). Thecalcium-binding protein S100A2 interacts with p53and modulates its transcriptional activity. J. Biol.Chem. 280, 29186–29193.
16. Koch, M., Bhattacharya, S., Kehl, T., Gimona, M.,Vasak, M., Chazin, W. et al. (2007). Implications onzinc binding to S100A2. Biochim. Biophys. Acta, 1773,457–470.
17. Randazzo, A., Acklin, C., Schäfer, B. W., Heizmann,C. W. & Chazin, W. J. (2001). Structural insight intohuman Zn2+-bound S100A2 from NMR and homol-ogy modeling. Biochem. Biophys. Res. Commun. 288,462–467.
18. Fritz, G., Mittl, P. R., Vasak, M., Grütter, M. G. &Heizmann, C. W. (2002). The crystal structure ofmetal-free human EF-hand protein S100A3 at 1.7-Åresolution. J. Biol. Chem. 277, 33092–33098.
19. Otterbein, L. R., Kordowska, J., Witte-Hoffmann, C.,Wang, C. L. & Dominguez, R. (2002). Crystalstructures of S100A6 in the Ca2+-free and Ca2+-bound states: the calcium sensor mechanism of S100proteins revealed at atomic resolution. Structure, 10,557–567.
20. Rustandi, R. R., Baldisseri, D. M., Inman, K. G.,Nizner, P., Hamilton, S. M., Landar, A. et al. (2002).Three-dimensional solution structure of the calcium-signaling protein apo-S100A1 as determined by NMR.Biochemistry, 41, 788–796.
21. Vallely, K. M., Rustandi, R. R., Ellis, K. C., Varlamova,O., Bresnick, A. R. & Weber, D. J. (2002). Solutionstructure of human Mts1 (S100A4) as determined byNMR spectroscopy. Biochemistry, 41, 12670–12680.
22. Maler, L., Potts, B. C. & Chazin, W. J. (1999). Highresolution solution structure of apo calcyclin andstructural variations in the S100 family of calcium-binding proteins. J. Biomol. NMR, 13, 233–247.
23. Dempsey, A. C., Walsh, M. P. & Shaw, G. S. (2003).Unmasking the annexin I interaction from thestructure of apo-S100A11. Structure, 11, 887–897.
24. Arnesano, F., Banci, L., Bertini, I., Fantoni, A., Tenori,L. & Viezzoli, M. S. (2005). Structural interplaybetween calcium(II) and copper(II) binding toS100A13 protein. Angew. Chem., Int. Ed. Engl. 44,6341–6344.
25. Drohat, A. C., Tjandra, N., Baldisseri, D. M. & Weber,D. J. (1999). The use of dipolar couplings fordetermining the solution structure of rat apo-S100B(ββ). Protein Sci. 8, 800–809.
26. Lee, Y. C., Volk, D. E., Thiviyanathan, V., Kleerekoper,Q., Gribenko, A. V., Zhang, S. et al. (2004). NMRstructure of the apo-S100P protein. J. Biomol. NMR, 29,399–402.
27. Skelton, N. J., Kordel, J. & Chazin, W. J. (1995).Determination of the solution structure of apocalbindin D9k by NMR spectroscopy. J. Mol. Biol.249, 441–462.
28. Fritz, G. & Heizmann, C. W. (2004). 3D structures ofthe calcium and zinc binding S100 proteins. In(Messerschmidt, A., Bode, W. & Cygler, M., eds),Handbook of Metalloproteins, Vol. 3, pp. 529–540JohnWiley and Sons, Inc., Chichester, England.
29. Koch, M., Diez, J. & Fritz, G. (2006). Purification andcrystallization of the human EF-hand tumour sup-pressor protein S100A2. Acta Crystallogr., Sect. F:Struct. Biol. Cryst. Commun. 62, 1120–1123.
30. Randazzo, A., Acklin, C., Schafer, B. W., Heizmann,C. W. & Chazin, W. J. (2001). Structural insight intohuman Zn2+-bound S100A2 from NMR and homologymodeling. Biochem. Biophys. Res. Commun. 288, 462–467.
31. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. &McCammon, J. A. (2001). Electrostatics of nanosys-tems: application to microtubules and the ribosome.Proc. Natl. Acad. Sci. USA, 98, 10037–10041.
32. Matsumura, H., Shiba, T., Inoue, T., Harada, S. & Kai,Y. (1998). A novel mode of target recognition sug-gested by the 2.0 Å structure of holo S100B frombovine brain. Structure, 6, 233–241.
942 Structure of Ca2+-Free S100A2
33. Brodersen, D. E., Nyborg, J. & Kjeldgaard, M. (1999).Zinc-binding site of an S100 protein revealed. Twocrystal structures of Ca2+-bound human psoriasin(S100A7) in the Zn2+-loaded and Zn2+-free states.Biochemistry, 38, 1695–1704.
34. Rety, S., Sopkova, J., Renouard, M., Osterloh, D.,Gerke, V., Tabaries, S. et al. (1999). The crystalstructure of a complex of p11 with the annexin II N-terminal peptide. Nat. Struct. Biol. 6, 89–95.
35. McClintock, K. A. & Shaw, G. S. (2003). A novel S100target conformation is revealed by the solutionstructure of the Ca2+–S100B–TRTK-12 complex. J.Biol. Chem. 278, 6251–6257.
36. Rintala-Dempsey, A. C., Santamaria-Kisiel, L., Liao,Y., Lajoie, G. & Shaw, G. S. (2006). Insights into S100target specificity examined by a new interactionbetween S100A11 and annexin A2. Biochemistry, 45,14695–14705.
37. Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I.,Tello, D., Dall'Acqua, W. et al. (1994). Bound watermolecules and conformational stabilization helpmediate an antigen–antibody association. Proc. Natl.Acad. Sci. USA, 91, 1089–1093.
38. Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W.& Richmond, T. J. (2002). Solvent mediated interac-tions in the structure of the nucleosome core particle at1.9 Å resolution. J. Mol. Biol. 319, 1097–1113.
39. Lu, Y., Wang, R., Yang, C. Y. & Wang, S. (2007).Analysis of ligand-bound water molecules in high-resolution crystal structures of protein–ligand com-plexes. J. Chem. Inf. Model. 47, 668–675.
40. Inman, K. G., Baldisseri, D. M., Miller, K. E. & Weber,D. J. (2001). Backbone dynamics of the calcium-signaling protein apo-S100B as determined by 15NNMR relaxation. Biochemistry, 40, 3439–3448.
41. Allen, F. H. (2002). The Cambridge Structural Data-base: a quarter of a million crystal structures andrising. Acta Crystallogr., Sect. B: Struct. Sci. 58, 380–388.
42. Harding, M. M. (2006). Small revisions to predicteddistances around metal sites in proteins. Acta Crystal-logr., Sect. D: Biol. Crystallogr. 62, 678–682.
43. Harding, M. M. (2002). Metal–ligand geometryrelevant to proteins and in proteins: sodium andpotassium. Acta Crystallogr., Sect. D: Biol. Crystallogr.58, 872–874.
44. Juers, D. H., Heightman, T. D., Vasella, A., McCarter,J. D., Mackenzie, L., Withers, S. G. & Matthews, B. W.(2001). A structural view of the action of Escherichiacoli (lacZ) beta-galactosidase. Biochemistry, 40,14781–14794.
45. Libson, A. M., Gittis, A. G., Collier, I. E., Marmer, B. L.,Goldberg, G. I. & Lattman, E. E. (1995). Crystalstructure of the haemopexin-like C-terminal domainof gelatinase A. Nat. Struct. Biol. 2, 938–942.
46. Faber,H. R., Groom,C. R., Baker,H.M.,Morgan,W. T.,Smith, A. & Baker, E. N. (1995). 1.8 Å crystal structureof the C-terminal domain of rabbit serum haemopexin.Structure, 3, 551–559.
47. Eberhard, M. & Erne, P. (1994). Calcium and magne-sium binding to rat parvalbumin. Eur. J. Biochem. 222,21–26.
48. Henzl, M. T., Larson, J. D. & Agah, S. (2004). Influenceof monovalent cation identity on parvalbumin diva-lent ion-binding properties. Biochemistry, 43, 2747–2763.
49. Skelton, N. J., Akke, M., Kordel, J., Thulin, E., Forsen,S. & Chazin, W. J. (1992). 15N NMR assignments and
chemical shift analysis of uniformly labeled 15Ncalbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states.FEBS Lett. 303, 136–140.
50. Skelton, N. J., Kordel, J., Forsen, S. & Chazin, W. J.(1990). Comparative structural analysis of the calciumfree and bound states of the calcium regulatoryprotein calbindin D9K. J. Mol. Biol. 213, 593–598.
51. Allouche, D., Parello, J. & Sanejouand, Y. H. (1999).Ca2+/Mg2+ exchange in parvalbumin and other EF-hand proteins. A theoretical study. J. Mol. Biol. 285,857–873.
52. Andersson, M., Malmendal, A., Linse, S., Ivarsson, I.,Forsén, S. & Svensson, L. A. (1997). Structural basis forthe negative allostery between Ca2+- and Mg2+-binding in the intracellular Ca2+-receptor calbindinD9K. Protein Sci. 6, 1139–1147.
53. Nayal, M. & Di Cera, E. (1996). Valence screening ofwater in protein crystals reveals potential Na+ bindingsites. J. Mol. Biol. 256, 228–234.
54. Stradal, T. B., Troxler, H., Heizmann, C. W. & Gimona,M. (2000). Mapping the zinc ligands of S100A2 by site-directed mutagenesis. J. Biol. Chem. 275, 13219–13227.
55. Diederichs, K. & Karplus, P. A. (1997). Improved R-factors for diffraction data analysis in macromolecularcrystallography. Nat. Struct. Biol. 4, 269–275.
56. Kabsch, W. (1993). Automatic processing of rotationdiffraction data from crystals of initially unknownsymmetry and cell constants. J. Appl. Crystallogr. 26,795–800.
57. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. &Read, R. J. (2005). Likelihood-enhanced fast transla-tion functions. Acta Crystallogr., Sect. D: Biol. Crystal-logr. 61, 458–464.
58. Storoni, L. C., McCoy, A. J. & Read, R. J. (2004).Likelihood-enhanced fast rotation functions. ActaCrystallogr., Sect. D: Biol. Crystallogr. 60, 432–438.
59. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997).Refinement of macromolecular structures by themaximum-likelihood method. Acta Crystallogr., Sect.D: Biol. Crystallogr. 53, 240–255.
60. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building proteinmodels in electron density maps and the location oferrors in these models. Acta Crystallogr., Sect. A: Found.Crystallogr. 47, 110–119.
61. Emsley, P. & Cowtan, K. (2004). Coot: model-buildingtools for molecular graphics. Acta Crystallogr., Sect. D:Biol. Crystallogr. 60, 2126–2132.
62. Kabsch, W. & Sander, C. (1983). Dictionary of proteinsecondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22,2577–2637.
63. Vaguine, A. A., Richelle, J. & Wodak, S. J. (1999).SFCHECK: a unified set of procedures for evaluatingthe quality of macromolecular structure-factor dataand their agreement with the atomic model. ActaCrystallogr., Sect. D: Biol. Crystallogr. 55, 191–205.
64. Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck stereochemical quality of protein structures. J.Appl. Crystallogr. 26, 283–291.
65. Yap, K. http://nmr.uhnres.utoronto.ca/ikura/interhlx/, University of Toronto.
66. Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G.,Mark, A. E. & Berendsen, H. J. (2005). GROMACS:fast, flexible, and free. J. Comput. Chem. 26, 1701–1718.
