The structure of rat mast cell protease II at 1.9-A resolution

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.     

The crystal structure of mercury-substituted poplar plastocyanin at 1.9-A resolution

The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(II)-plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(II) atom in plastocyanin by Hg(II) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(II)-plastocyanin remains weak in Hg(II)-plastocyanin. The "flip" of a proline side chain close to the metal site from a C gamma-exo conformation in Cu(II)-plastocyanin to a C gamma-endo conformation in Hg(II)-plastocyanin suggests that this region of the molecule is particularly flexible. Crystallographic evidence for the close similarity of the Hg(II)- and Cu(II)-plastocyanin structures was originally obtained from electron density difference maps at 2.5-A resolution. The refinement of the structure was begun with a set of atomic coordinates taken from the structure of Cu(II)-plastocyanin. A Hg(II) atom was substituted for the Cu(II) atom, and the side chains of 6 residues in the vicinity of the metal site were omitted. Three series of stereochemically restrained least-squares refinement calculations were interspersed with two stages of model adjustment followed by phase extension. Fifty-nine water molecules were located. The final structure has a crystallographic residual R = 0.16.     

The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution.

The three-dimensional structure of recombinant human lymphotoxin (residues 24-171 of the mature protein) has been determined by x-ray crystallography at 1.9-A resolution (Rcryst = 0.215 for I greater than 3 sigma (I)). Phases were derived by molecular replacement using tumor necrosis factor (TNF-alpha) as a search model. Like TNF-alpha, lymphotoxin (LT) folds to form a "jellyroll" beta-sheet sandwich. Three-fold related LT subunits form a trimer stabilized primarily by hydrophobic interactions. A cluster of 6 basic residues around the 3-fold axis may account for the acid lability of the trimer. Although the structural cores of TNF-alpha and LT are similar, insertions and deletions relative to TNF-alpha occur in loops at the "top" of the LT trimer and significantly alter the local structure and the overall shape trimer is highly conserved. The sites of two mutations (Asp-50 and Tyr-108) that abolish the cytotoxicity of LT are contained within poorly ordered loops of polypeptide chain that flank the cleft between neighboring subunits at the base of the molecule, suggesting that the receptor recognizes an intersubunit binding site.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.     

Structure of yeast triosephosphate isomerase at 1.9-A resolution

The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0%. The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein. The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96. In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed. For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact. Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers. The analysis also suggests that the interface may be a particularly good target for drug design. The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure.     

Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.     

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Zymomonas mobilis at 1.9-A resolution

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-A resolution, and that of a binary complex with the co-substrate NADPH to 2.7-A resolution. The subunit of DXR consists of three domains. Residues 1-150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed beta-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300-386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after beta1 and with the loops between alpha2 and beta2 and alpha5 and beta5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.     

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.     

Refined atomic model of wheat serine carboxypeptidase II at 2.2-A resolution.

The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-A resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 A and from bond angles of 2.8 degrees. The model supports the general conclusions of an earlier study at 3.5-A resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha + beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad" Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity. Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular structure of an apolipoprotein determined at 2.5-A resolution

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.     

Crystal structure of alpha-hordothionin at 1.9 Angstrom resolution

Crystal structure of ubiquitous toxin from barley alpha-hordothionin (alpha-HT) has been determined at 1.9A resolution by X-ray crystallography. The primary sequence as well as the NMR solution structure of alpha-HT firmly established that alpha-HT belongs to a family of membrane active plant toxins-thionins. Since alpha-HT crystallized in a space group (P4(1)2(1)2) that is different from the space group (I422) of previously determined alpha(1)- and beta-purothionins, and visocotoxin A3, therefore, it provided independent information on protein-protein interactions that may be relevant to the toxin mechanism. The structure of alpha-HT not only confirms overall architectural features (crambin fold) but also provides an additional confirmation of the role for crucial solute molecules, that were postulated to be directly involved in the mechanism of toxicity for thionins.     

Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9A resolution

Even being a bacterial purine nucleoside phosphorylase (PNP), which normally shows hexameric folding, the Mycobacterium tuberculosis PNP (MtPNP) resembles the mammalian trimeric structure. The crystal structure of the MtPNP apoenzyme was solved at 1.9 A resolution. The present work describes the first structure of MtPNP in complex with phosphate. In order to develop new insights into the rational drug design, conformational changes were profoundly analyzed and discussed. Comparisons over the binding sites were specially studied to improve the discussion about the selectivity of potential new drugs.     

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase

The structure of serine carboxypeptidase II from wheat bran has been determined to 3.5-A resolution by multiple isomorphous replacement, solvent flattening, and crystallographic refinement. The amino acid sequence has been fit to the electron density map and the model refined to a conventional crystallographic R factor of 20.9%. The molecule is an alpha + beta protein and contains a "catalytic triad" (Asp338, His397, and Ser146) similar in arrangement to those in chymotrypsin and subtilisin. The -fold of the polypeptide backbone is, however, completely different from those enzymes. This suggests that this is a third example of convergent evolution to a common enzymatic mechanism. The -fold is, on the other hand, surprisingly similar to that of the zinc proteinase carboxypeptidase A.     

The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution.

The crystal and molecular structure of a triacylglyceride lipase (EC 3.1.1.3) from the fungus Rhizomucor miehei was analyzed using X-ray single crystal diffraction data to 1.9 A resolution. The structure was refined to an R-factor of 0.169 for all available data. The details of the molecular architecture and the crystal structure of the enzyme are described. A single polypeptide chain of 269 residues is folded into a rather unusual singly wound beta-sheet domain with predominantly parallel strands, connected by a variety of hairpins, loops and helical segments. All the loops are right-handed, creating an uncommon situation in which the central sheet is asymmetric in that all the connecting fragments are located on one side of the sheet. A single N-terminal alpha-helix provides the support for the other, distal, side of the sheet. Three disulfide bonds (residues 29-268, 40-43, 235-244) stabilize the molecule. There are four cis peptide bonds, all of which precede proline residues. In all, 230 ordered water molecules have been identified; 12 of them have a distinct internal character. The catalytic center of the enzyme is made up of a constellation of three residues (His257, Asp203 and Ser144) similar in structure and function to the analogous (but not homologous) triad found in both of the known families of serine proteinases. The fourth residue in this system equivalent to Thr/Ser in proteinases), hydrogen bonded to Asp, is Tyr260. The catalytic site is concealed under a short amphipatic helix (residues 85 to 91), which acts as "lid", opening the active site when the enzyme is adsorbed at the oil-water interface. In the native enzyme the "lid" is held in place by hydrophobic interactions.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

The structure of prothrombin fragment 1 at 3.5-A resolution

The structure of prothrombin fragment 1 has been determined at 3.5-A resolution by multiple isomorphous replacement methods with four heavy atom derivatives. The final average figure of merit is 0.72. There is a large cylindrical solvent region with an average diameter of 35-40 A along the entire length of the c axis (85 A) centered at about x = y = 1/2. The connected density forming the wall of this channel is not of sufficient extent to account for the 156 residues of fragment 1 and the two accompanying carbohydrate chains totaling 5000 in molecular weight. Deglycosylated fragment 1 crystallizes isomorphously with fragment 1, and a difference map between the two revealed that the sugar chains are severely disordered and reside in the solvent channel. Although the disordered carbohydrate and the complexity of five disulfides in a 126-residue sequence have hampered the complete tracing of the peptide chain, two-thirds of the molecule has been accounted for in the form of an unusually oblate ellipsoid of about 15 X 30 X 35 A. The folding of the molecule has little secondary structure (one alpha-helix (7%), 20% beta-structure) in agreement with dichroism measurements and one of the points of carbohydrate attachment is suggested from the deglycosylated difference map.     

The crystal structure of erabutoxin a at 2.0-A resolution

The three-dimensional structure of erabutoxin a, a single-chain, 62-residue protein neurotoxin from snake venom, has been determined to 2.0-A resolution by x-ray crystal structure analysis. Molecular replacement methods were used, and the structure refined to a residual R = 0.17. The sites of 62 water molecules and 1 sulfate ion have been located and refined. The structure of erabutoxin a is very similar to that established earlier for erabutoxin b. These toxins from venom of the same snake differ in sequence only at residue 26, which is Asn in erabutoxin a and His in erabutoxin b. The substitution leads to only minor variations in intramolecular hydrogen bonding. Furthermore, the distribution of thermal parameters and the implied regional mobilities are similar in the two structures. In particular, the highly mobile character of the peripheral segment Pro44-Gly49 in both structures supports the specific role proposed for this segment in neurotoxin binding to the acetylcholine receptor. Forty-eight of the solvent sites determined are first surface positions; approximately one-half of these are equivalent to solvent sites in erabutoxin b.     

Crystal structure of Ca2+ -free S100A2 at 1.6-A resolution

S100A2 is an EF hand-containing Ca^2 +-binding protein of the family of S100 proteins. The protein is localized exclusively in the nucleus and is involved in cell cycle regulation. It attracted most interest by its function as a tumor suppressor via p53 interaction. We determined the crystal structure of homodimeric S100A2 in the Ca^2 +-free state at 1.6-Å resolution. The structure revealed structural differences between subunits A and B, especially in the conformation of a loop that connects the N- and C-terminal EF hands and represents a part of the target-binding site in S100 proteins. Analysis of the hydrogen bonding network and molecular dynamics calculations indicate that one of the two observed conformations is more stable. The structure revealed Na⁺ bound to each N-terminal EF hand of both subunits coordinated by oxygen atoms of the backbone carbonyl and water molecules. Comparison with the structures of Ca^2 +-free S100A3 and S100A6 suggests that Na⁺ might occupy the S100-specific EF hand in the Ca^2 +-free state.