Similar active sites in lysostaphins and D-Ala-D-Ala metallopeptidases

Specific peptidases exist for nearly every amide linkage in peptidoglycan. In several cases, families of peptidoglycan hydrolases with different specificities turned out to be related. Here we show that lysostaphin-type peptidases and D-Ala-D-Ala metallopeptidases have similar active sites and share a core folding motif in otherwise highly divergent folds. The central Zn(2+) is tetrahedrally coordinated by two histidines, an aspartate, and a water molecule. The Zn(2+) chelating residues occur in the order histidine, aspartate, histidine in all sequences and contact the metal via the Nepsilon, the Odelta, and the Ndelta, respectively. The identity of the other active-site residues varies, but in all enzymes of known structure except for VanX, a conserved histidine is present two residues upstream of the second histidine ligand to the Zn(2+). As the same arrangement of active-site residues is also found in the N-terminal, cryptic peptidase domain of sonic hedgehog, we propose that this arrangement of active-site residues be called the "LAS" arrangement, because it is present in lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and in the cryptic peptidase in the N-domain of sonic hedgehog.     

Crystal structures of active LytM

Lysostaphin-type enzymes are metalloendopeptidases that are present in bacteriophages and in bacteria. They share the catalytic domain, but normally contain other domains as well. The well-characterized enzymes in this group are all specific for the pentaglycine crosslinks in the cell walls of some Gram-positive bacterial species. Lysostaphin-type enzymes are synthesized as secreted preproenzymes and require proteolytic activation for maturation. Although lysostaphin, the prototypical peptidase in the group, is widely used as a tool in biotechnology and developed as an antistaphylococcal agent, the detailed structure of this enzyme is unknown. So far, only one lysostaphin-type enzyme, the Staphylococcus aureus autolysin LytM, has been crystallized in its full-length, inactive form. Here, we describe the synthesis of a convenient reporter substrate, characterize the metal and pH-dependence of an active LytM fragment, and present its crystal structure in three crystal forms at different pH values that either support or do not support activity. In all structures, we find an extended, long and narrow groove that has the active site at its bottom and is delineated on the sides by the most flexible regions of the molecule. In two cases, the groove is partially filled by a loop of a neighbouring molecule in the crystal. As the loop contains three consecutive glycine residues, this crystal packing effect supports the interpretation that the groove is the substrate-binding cleft. To characterize the substrate-binding mode more closely, a phosphinate analogue of tetraglycine was synthesized. Although tetraglycine is a substrate of the active LytM fragment, the phosphinate analogue turned out to be a very poor inhibitor. Crystals that were grown in its presence contained an L+-tartrate molecule from the crystallization buffer and not the phosphinate in the active site.     

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.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.     

Alternating zinc fingers in the human male-associated protein ZFY: HX3H and HX4H motifs encode a local structural switch

The two-finger repeat in the human male-associated protein ZFY provides a model for comparative 2D-NMR studies of classical and variant Zn fingers. This repeat is defined in part by an alternation in spacing between consensus (HX3H) and variant (HX4H) histidine spacings. To investigate the effects of a "switch" between alternative histidine spacings, we have designed an HX3H analogue of a representative HX4H domain of known structure [ZFY-6; Kochoyan, M., Havel, T., Nguyen, D. T., Dahl, C. E., Keutmann, H. T., & Weiss, M. A. (1991) Biochemistry 30, 3371-3386]. The HX3H analogue (designated ZFY-switch) forms a tetrahedral Co2+ complex whose thermodynamic stability is similar to that of the parent peptide. 2D-NMR studies demonstrate that ZFY-switch and ZFY-6, although similar in overall structure, exhibit significant local changes near the site of deletion. Whereas the HX4H site in the native finger forms a nonstandard loop, the HX3H site in ZFY-switch folds as a 3(10) extension of the C-terminal alpha-helix, as observed in the NMR solution structure of a consensus HX3H domain [Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A., & Wright, P. E. (1989) Science 245, 635-637] and in the crystal structure of a representative Zn finger-DNA complex [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. We propose that variant histidine spacings (HX3H and HX4H) encode a local switch between alternative surface architectures with implications for models of protein-DNA recognition.     

Cloning,overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.     

The metal binding site of zoocin A

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site.On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2 +/- 0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.     

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.     

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.     

Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn(2+)-bound and Mn(2+)-bound complexes in the presence of phosphate ions at resolutions of 2.6 A to 2.0 A. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel beta-strands linked to a C-terminal alpha-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg(2+)-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn(2+) or Mn(2+) ions were observed in the center of the H-N-H motif in cases of Zn(2+) or Mn(2+)-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.     

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

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.     

Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites

Previously, we have identified three Zn(2+) binding residues in an endogenous Zn(2+) binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn(2+) binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn(2+)-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn(2+) potently inhibited [(3)H]dopamine uptake with an IC(50) value of 7 microM as compared to a value of 300 microM for the control (H193K). These data are consistent with the presence of an alpha-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn(2+) affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [(3)H]dopamine uptake by Zn(2+) (IC(50) = 3-32 microM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn(2+) binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.     

Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.     

Mutational analysis of the active site of human insulin-regulated aminopeptidase.

Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn(2+)-coordination sequence element, HEXXH-(18-64X)-E. A second conserved sequence element, the GXMEN motif, positioned 22-32 amino acids N-terminal to the Zn(2+)-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full-length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn(2+)-binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine-para-nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn(2+)-binding motifs is important for IRAP enzyme activity.     

The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases

The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, using fluorescence resonance energy transfer (FRET) assays, that the DNA hydrolysis activity of H545 N-ColE7 mutants was completely abolished while activities of N560 and H573 mutants varied from 6.9% to 83.2% of the wild-type activity. The crystal structures of three N-ColE7 mutants in complex with the inhibitor Im7, N560A-Im7, N560D-Im7 and H573A-Im7, were determined at a resolution of 1.9 A to 2.2 A. H573 is responsible for metal ion binding in the wild-type protein, as the zinc ion is still partially associated in the structure of H573A, suggesting that H573 plays a supportive role in metal binding. Both N560A and N560D contain a disordered loop in the HNH motif due to the disruption of the hydrogen bond network surrounding the side-chain of residue 560, and as a result, the imidazole ring of the general base residue H545 is tilted slightly and the scissile phosphate is shifted, leading to the large reductions in hydrolysis activities. These results suggest that the highly conserved asparagine in the HNH motif, in general, plays a structural role in constraining the loop in the metal finger structure and keeping the general base histidine and scissile phosphate in the correct position for DNA hydrolysis.     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.     

R1 in the Shaker S4 occupies the gating charge transfer center in the resting state

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1-R4) cross the transmembrane electric field. It has been proposed that R1-R4 movement is facilitated by a "gating charge transfer center" comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn(2+) on channel activity. In I287H+R1H, Zn(2+) generated a slow component of activation with a maximum amplitude (A(slow,max)) of ～56%, indicating that only a fraction of voltage sensors can bind Zn(2+) at a holding potential of -80 mV. A(slow,max) decreased after applying either depolarizing or hyperpolarizing prepulses from -80 mV. The decline of A(slow,max) after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3-S4 loop generated a Zn(2+)-binding site. At saturating concentrations, A(slow,max) reached 100%, indicating that Zn(2+) traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn(2+) binding at -80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.