Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase

Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity. Amino acid substitution of His168 resulted in a drastic decrease in the enzyme's catalytic activity, consistent with its putative catalytic role. Substitutions were also carried out in neighboring residues, Lys166, Asp167, and Gln169, in an attempt to alter the enzyme's substrate binding site. Lys166 and Gln169 mutants showed only minor changes in substrate specificity profiles. In sharp contrast, mutants of Asp167 showed considerably altered specificity profiles. Of the numerous Asp167 mutants characterized, Asp167Glu showed the best substrate specificity profile, while retaining most of its catalytic activity for glucose and stability. We also investigated the cumulative effect of combining the Asp167Glu substitution with the previously reported Asn452Thr mutation. Interpretation of the effect of the replacement of Asp167 to Glu on the alteration of substrate specificity in relation with the predicted 3D model of PQQGDH-B is also discussed.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

A helical turn motif in Mss4 is a critical determinant of Rab binding and nucleotide release

Monomeric Rab GTPases function as ubiquitous regulators of intracellular membrane trafficking. Mss4, an evolutionarily conserved Rab accessory factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Here we describe the results of a high-resolution crystallographic and mutational analysis of Mss4. The 1.65 A crystal structure of Mss4 reveals a network of direct and water-mediated interactions that stabilize a partially exposed structural subdomain derived from four highly conserved but nonconsecutive sequence elements. The conserved subdomain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DPhiPhi motif, consisting of an invariant aspartic acid residue (Asp 73) followed by two bulky hydrophobic residues (Met 74 and Phe 75), encodes a prominently exposed 3(10) helical turn in which the backbone is well-ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alanine dramatically impairs nucleotide release activity toward Rab3A, indicating that the DPhiPhi motif is a critical element of the Rab interaction epitope. In particular, mutation of Phe 75 results in a defect as severe as that observed for mutation of Asp 96, which is located near the zinc binding site at the opposite end of the conserved subdomain. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate nucleotide release.     

A zinc-binding region in Vif binds Cul5 and determines cullin selection

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.     

Critical role of transmembrane segment zinc binding in the structure and function of rhodopsin

Zinc deficiency and retinitis pigmentosa are both important factors resulting in retinal dysfunction and night blindness. In this study, we address the critical biochemical and structural relevance of zinc ions in rhodopsin and examine whether zinc deficiency can lead to rhodopsin dysfunction. We report the identification of a high-affinity zinc coordination site within the transmembrane domain of rhodopsin, coordinated by the side chains of two highly conserved residues, Glu(122) in transmembrane helix III and His(211) in transmembrane helix V. We also demonstrate that this zinc coordination is critical for rhodopsin folding, 11-cis-retinal binding, and the stability of the chromophore-receptor interaction, defects of which are observed in retinitis pigmentosa. Furthermore, a cluster of retinitis pigmentosa mutations is localized within and around this zinc binding site. Based on these studies, we believe that improvement in zinc binding to rhodopsin at this site within the transmembrane domain may be a pharmacological approach for the treatment of select retinitis pigmentosa mutations. Transmembrane coordination of zinc may also be an important common principle across G-protein-coupled receptors.     

Human glutathione-dependent formaldehyde dehydrogenase. Structural changes associated with ternary complex formation

Human glutathione-dependent formaldehyde dehydrogenase plays an important role in the metabolism of glutathione adducts such as S-(hydroxymethyl)glutathione and S-nitrosoglutathione. The role of specific active site residues in binding these physiologically important substrates and the structural changes during the catalytic cycle of glutathione-dependent formaldehyde dehydrogenase was examined by determining the crystal structure of a ternary complex with S-(hydroxymethyl)glutathione and the reduced coenzyme to 2.6 A resolution. The formation of the ternary complex caused the movement of the catalytic domain toward the coenzyme-binding domain. This represents the first observation of domain closure in glutathione-dependent formaldehyde dehydrogenase in response to substrate binding. A water molecule adjacent to the 2'-ribose hydroxyl of NADH suggests that the alcohol proton is relayed to solvent directly from the coenzyme, rather than through the action of the terminal histidine residue as observed in the proton relay system for class I alcohol dehydrogenases. S-(Hydroxymethyl)glutathione is directly coordinated to the active site zinc and forms interactions with the highly conserved residues Arg114, Asp55, Glu57, and Thr46. The active site zinc has a tetrahedral coordination environment with Cys44, His66, and Cys173 as the three protein ligands in addition to S-(hydroxymethyl)glutathione. This is in contrast to zinc coordination in the binary coenzyme complex where all of the ligands were contributed by the enzyme and included Glu67 as the fourth protein ligand. This change in zinc coordination is accomplished by an approximately 2.3 A movement of the catalytic zinc.     

Structure/function analyses of human sex hormone-binding globulin: effects of zinc on steroid-binding specificity

In humans, sex hormone-binding globulin (SHBG) binds and transports the biologically most important androgens and estrogens in the blood, and regulates the access of these steroids to their targets tissues. In addition to binding sex steroids, SHBG has specific binding sites for divalent cations including calcium and zinc. Zinc binding to a site at the entrance of the steroid-binding pocket in human SHBG has been shown to reduce its affinity for estrogens, while having no impact on the binding of C19 steroids. Crystallographic studies indicate that C18 and C19 steroids are bound in opposite orientations within the SHBG steroid-binding site, and we have obtained new information that supports a molecular model explaining the mechanism by which zinc alters the affinity of human SHBG for estrogens, by studying directly the estradiol-binding properties SHBG variants created by site-directed mutagenesis. In this model, the coordination of a zinc ion by the side chains of residues Asp65 and His136 eliminates a critical hydrogen bond between Asp65 and the hydroxyl at C3 of estrogens, such as estradiol and 2-methoxyestradiol, and causes disorder in a polypeptide loop segment that covers the steroid-binding site. The combination of these structural changes explains the specific decrease in the affinity of human SHBG for C18 steroids in the presence of a zinc ion.     

X-Ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes

Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge x-ray absorption fine-structure spectroscopy, we report here on the local structure of Zn2+ bound stoichiometrically to noncrystallized cyt bc1 complexes. We performed a comparative x-ray absorption fine-structure spectroscopy study by examining avian, bovine, and bacterial enzymes. A large number of putative clusters, built by combining information from first-shell analysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme, a tetrahedral ligand cluster formed by two His, one Lys, and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme, the ligands were the His121, His268, Lys270, and Asp253 residues, and in the homologous bovine enzyme they were the His121, His267, Lys269, and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of 6. The best-fit octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue, and two water molecules. It was interesting that by aligning the crystallographic structures of the bacterial and avian enzymes, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295, and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donors/acceptors.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.     

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence,conserved residues,and proton hyperfine structure

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.     

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.     

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.     

A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis

QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Binding studies with mutants of Zif268. Contribution of individual side chains to binding affinity and specificity in the Zif268 zinc finger-DNA complex.

The Zif268 zinc finger-DNA complex has served as a model system for understanding how Cys2His2 type zinc fingers recognize DNA. Structural studies of the Zif268-DNA complex revealed that residues at four positions in the alpha helix of each zinc finger play key roles in recognition, but there has been no information about the precise contributions of individual residues. Here we report the results of binding studies involving five mutants of Zif268 that have changes in the base-contacting residues of finger one. These studies let us evaluate the contributions that Arg18 (position -1 of the alpha helix), Asp20 (position 2), Glu21 (position 3), and Arg24 (position 6) make to the overall energy of DNA binding. Our results confirm the important role played by these arginines. By comparing the affinities of the wild type and mutant peptides for various sites, we also prove that Asp20 and Glu21 play important roles in determining binding site specificity.     

Sequence variability analysis of human class I and class II MHC molecules: functional and structural correlates of amino acid polymorphisms

Major histocompatibility complex class I (MHCI) and class II (MHCII) molecules display peptides on antigen-presenting cell surfaces for subsequent T-cell recognition. Within the human population, allelic variation among the classical MHCI and II gene products is the basis for differential peptide binding, thymic repertoire bias and allograft rejection. While available 3D structural analysis suggests that polymorphisms are found primarily within the peptide-binding site, a broader informatic approach pinpointing functional polymorphisms relevant for immune recognition is currently lacking. To this end, we have now analyzed known human class I (774) and class II (485) alleles at each amino acid position using a variability metric (V). Polymorphisms (V>1) have been identified in residues that contact the peptide and/or T-cell receptor (TCR). Using sequence logos to investigate TCR contact sites on HLA molecules, we have identified conserved MHCI residues distinct from those of conserved MHCII residues. In addition, specific class II (HLA-DP, -DQ, -DR) and class I (HLA-A, -B, -C) contacts for TCR binding are revealed. We discuss these findings in the context of TCR restriction and alloreactivity.     

Identification of essential catalytic residues of the cyclase NisC involved in the biosynthesis of nisin

Nisin is a post-translationally modified antimicrobial peptide that has been widely used in the food industry for several decades. It contains five cyclic thioether cross-links of varying sizes that are installed by a single enzyme, NisC, that catalyzes the addition of cysteines to dehydroamino acids. The recent x-ray crystal structure of NisC has provided the first insights into the catalytic residues responsible for the cyclization step during nisin biosynthesis. In this study, the conserved residues His(212), Arg(280), Asp(141), and Tyr(285) as well as the ligands to the zinc in the active site (Cys(284), Cys(330), and His(331)) were substituted by site-directed mutagenesis. Binding studies showed that all mutants had similar affinities for NisA. Activity assays showed that whereas His(212) and Asp(141) were essential for correct cyclization as judged by the antimicrobial activity of the final product, Arg(280) and Tyr(285) were not. Mutation of zinc ligands to alanine also abolished the enzymatic activity, and these mutant proteins were shown to contain decreased levels of zinc. These results show that the zinc is essential for activity and support a model in which the zinc is used to activate the cysteines in the substrate for nucleophilic attack. These findings also argue against an essential role of Arg(280) and Tyr(285) as an active site general acid/base in the mechanism of cyclization.     

Essential features of the catalytic core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase

Bioactive peptides frequently terminate with an essential alpha-amide that is generated from a COOH-terminal Gly in a two-step enzymatic process occurring within the lumen of the secretory pathway. The first enzyme, peptidylglycine alpha-hydroxylating monooxygenase, is a member of the copper- and ascorbate-dependent monooxygenase family. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL, EC 4.3.2.5), has no known homologues. Examination of the catalytic core of PAL (PALcc) using trypsin, BNPS skatole, and COOH-terminally truncated proteins failed to identify stable subdomains. Treatment of PALcc with divalent metal ion chelators inactivated the enzyme and increased its protease and thermal sensitivity, suggesting a structural role for bound metal. Purified PALcc contained 0.7 +/- 0.4 mol of zinc/mol of enzyme. Since the four Cys residues in PALcc form two disulfide bonds, potential Zn ligands include conserved Asp, Glu, and His residues. The secretion and activity of PALcc bearing mutations in each conserved Asp, Glu, and His residue were evaluated. Mutation of three conserved Asp residues and two conserved His residues yielded a protein that could not be secreted, suggesting that these residues play a structural role. Analysis of mutants that were efficiently secreted identified three His residues along with single Asp residue that may play a role in catalysis. These essential residues occur in a pattern unique to PAL.