Peptidoglycan amidase MepA is a LAS metallopeptidase

LAS enzymes are a group of metallopeptidases that share an active site architecture and a core folding motif and have been named according to the group members lysostaphin, D-Ala-D-Ala carboxypeptidase and sonic hedgehog. Escherichia coli MepA is a periplasmic, penicillin-insensitive murein endopeptidase that cleaves the D-alanyl-meso-2,6-diamino-pimelyl amide bond in E. coli peptidoglycan. The enzyme lacks sequence similarity with other peptidases, and is currently classified as a peptidase of unknown fold and catalytic class in all major data bases. Here, we build on our observation that two motifs, characteristic of the newly described LAS group of metallopeptidases, are conserved in MepA-type sequences. We demonstrate that recombinant E. coli MepA is sensitive to metal chelators and that mutations in the predicted Zn2+ ligands His-113, Asp-120, and His-211 inactivate the enzyme. Moreover, we present the crystal structure of MepA. The active site of the enzyme is most similar to the active sites of lysostaphin and D-Ala-D-Ala carboxypeptidase, and the fold is most closely related to the N-domain of sonic hedgehog. We conclude that MepA-type peptidases are LAS enzymes.     

Latent LytM at 1.3A resolution

LytM, an autolysin from Staphylococcus aureus, is a Zn(2+)-dependent glycyl-glycine endopeptidase with a characteristic HxH motif that belongs to the lysostaphin-type (MEROPS M23/37) of metallopeptidases. Here, we present the 1.3A crystal structure of LytM, the first structure of a lysostaphin-type peptidase. In the LytM structure, the Zn(2+) is tetrahedrally coordinated by the side-chains of N117, H210, D214 and H293, the second histidine of the HxH motif. Although close to the active-site, H291, the first histidine of the HxH motif, is not directly involved in Zn(2+)-coordination, and there is no water molecule in the coordination sphere of the Zn(2+), suggesting that the crystal structure shows a latent form of the enzyme. Although LytM has not previously been considered as a proenzyme, we show that a truncated version of LytM that lacks the N-terminal part with the poorly conserved Zn(2+) ligand N117 has much higher specific activity than full-length enzyme. This observation is consistent with the known removal of profragments in other lysostaphin-type proteins and with a prior observation of an active LytM degradation fragment in S.aureus supernatant. The "asparagine switch" in LytM is analogous to the "cysteine switch" in pro-matrix metalloproteases.     

Mutational analysis of peptidoglycan amidase MepA

Murein endopeptidase A (MepA) from Escherichia coli is a periplasmic peptidoglycan amidase that cleaves d,d amide bonds between d-alanine and meso-2,6-diaminopimelic acid in E. coli peptidoglycan. MepA and its homologues in other proteobacteria share overall structural similarity with d-Ala-d-Ala metallopeptidases and local similarity around the active site with lysostaphin-type enzymes, which has prompted the classification of these enzymes as LAS enzymes. LAS enzymes contain a single divalent cation in the active site, which is tetracoordinated in the crystal structures. Three of the metal ligands are identical in all structures, but the identity of the fourth ligand varies. Two residues in proximity to the metal might act as a general acid/base, but their role is not clear. Here, we report a new MepA expression system, which allows the separation of MepA variants from the endogenous wild-type enzyme, and an HPLC assay with a defined peptidoglycan fragment, which allows assessment of MepA activity without a refolding step. We find that the conserved metal ligands are required for folding (D120) or catalysis (H113, H211). Separate mutations of the candidate catalytic residues H206 or H209 and of the "fourth" metal ligand H110 are tolerated for folding but drastically reduce activity. Mutation of residue W203 to aspartate impairs substrate binding.     

Bacillus subtilis CwlP of the SP-{beta} prophage has two novel peptidoglycan hydrolase domains, muramidase and cross-linkage digesting DD-endopeptidase

For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.     

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (beta subunits) from several vertebrates (47-52% and 50-60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.     

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.     

Sequence homologies between argininosuccinase, aspartase and fumarase: A family of structurally-related enzymes

Abstract Aspartase, fumarase (Class II) and argininosuccinase are homotetrameric enzymes of native M _r∼ 200 000, and they all catalyse analogous trans -elimination reactions involving fumarate. Amino acid sequence comparisons have revealed five mutually-conserved segments of primary structure, indicating that the enzymes belong to the same family. Several putative active-site residues, e.g., histidine, methionine, lysine, and aspartate or glutamate, have been detected, and one of the highly-conserved segments was also found in an otherwise unrelated homodimeric Class I fumarase.     

Unraveling the substrate-metal binding site of ferrochelatase: an X-ray absorption spectroscopic study

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)(3)(S/Cl)(1) coordination for the yeast and Zn(N/O)(2)(S/Cl)(2) coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co(2+) coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co(2+), Fe(2+) bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.     

Amidase domains from bacterial and phage autolysins define a family of gamma-D,L-glutamate-specific amidohydrolases

Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (N-acetylmuramoyl-L-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-D-glutamyl-L-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage lambda. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.     

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc.     

Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes.     

Structure-based analysis of the metal-dependent mechanism of H-N-H endonucleases

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.     

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction.     

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.     

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.