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1.
2.
A zinc-binding region in Vif binds Cul5 and determines cullin selection   总被引:1,自引:0,他引:1  
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.  相似文献   

3.
Lacombe T  Gabriel JM 《FEBS letters》2002,531(3):469-474
The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.  相似文献   

4.
Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.  相似文献   

5.
6.
The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.  相似文献   

7.
The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.  相似文献   

8.
Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge number of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate experimentally. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiological function. This is achieved by a combination of approaches, which include: (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protein domains based on multiple sequence alignments of known metalloproteins obtained from the Pfam database; and (iii) mining the annotations of human gene sequences, which are based on any type of information available. It is found that 1684 proteins in the human proteome are independently identified by all three approaches as zinc-proteins, 746 are identified by two, and 777 are identified by only one method. By assuming that all proteins identified by at least two approaches are truly zinc-binding and inspecting the proteins identified by a single method, it can be proposed that ca. 2800 human proteins are potentially zinc-binding in vivo, corresponding to 10% of the human proteome, with an uncertainty of 400 sequences. Available functional information suggests that the large majority of human zinc-binding proteins are involved in the regulation of gene expression. The most abundant class of zinc-binding proteins in humans is that of zinc-fingers, with Cys4 and Cys2His2 being the most common types of coordination environment.  相似文献   

9.
The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. AVAILABILITY: Freely available at http://metaldetector.dsi.unifi.it. SUPPLEMENTARY INFORMATION: Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.  相似文献   

10.
11.
S Geeganage  P A Frey 《Biochemistry》1999,38(40):13398-13406
Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mol of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 A from His 166 in active site, and the iron site lies 30 A from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that C52S-, C55S-, and H164N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1,10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential.  相似文献   

12.
The crystal structure of a putative protease from Bacteroides thetaiotaomicron (ppBat) suggested the presence of a zinc ion in each protomer of the dimer as well as a flavin in the dimer interface. Since the chemical identity of the flavin and the exact mode of binding remained unclear, we have determined the crystal structure of ppBat in complex with riboflavin. The obtained structure revealed that the isoalloxazine ring is sandwiched between two tryptophan residues (Trp164) from both chains and adopts two alternate orientations with the N(10)-ribityl side chain protruding from the binding site in opposite directions. In order to characterize the zinc-binding site, we generated two single variants and one double variant in which the two coordinating cysteine residues (Cys74 and Cys111) were replaced by alanine. All three variants were unable to bind zinc demonstrating that both cysteine residues are essential for binding. Moreover, the lack of zinc binding also resulted in drastically reduced thermal stability (11–15 °C). A similar effect was obtained when wild-type protein was incubated with EDTA supporting the conclusion that the zinc-binding site plays an important structural role in ppBat. On the other hand, attempts to identify proteolytic activity failed suggesting that the zinc may not act as a catalytic center in ppBat. Structurally similar zinc binding motives in other proteins were also found to play a structural rather than catalytic role and hence it appears that neither the flavin nor the zinc binding sites possess a catalytic function in ppBat.  相似文献   

13.
Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237-->Ala and Cys239-->Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding.  相似文献   

14.
The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.  相似文献   

15.
Histidines 107 and 109 in the glycine receptor (GlyR) alpha1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing alpha1 subunits containing the H107A mutation with alpha1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha1 subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha1beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha1 homomers. The binding of zinc at the interface between adjacent alpha1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.  相似文献   

16.
Sequence variation in transcription factor IIIA   总被引:5,自引:2,他引:3       下载免费PDF全文
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17.
The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.  相似文献   

18.
Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.  相似文献   

19.
Adamczyk M  Poznański J  Kopera E  Bal W 《FEBS letters》2007,581(7):1409-1416
UV spectroscopy demonstrated that chicken mononucleosomes bind Co(II) and Zn(II) ions at submicromolar concentrations in a tetrahedral mode, at a conserved zinc finger-like site, composed of Cys110 and His113 residues of both H3 molecules. Neither of these metal ions substituted for another, indicating a limited binding reversibility. Molecular modeling indicated that the tetrahedral site is formed by unhindered rotations around Calpha-Cbeta bonds in the side chains of the zinc binding residues. The resulting local rearrangement of the protein structure shields the bound metal ion from the solvent, explaining the observed lack of reversibility of the binding. Consequences of these findings for zinc homeostasis, metal toxicology and nucleosomal regulation are discussed.  相似文献   

20.
Yu C  Zavaljevski N  Desai V  Reifman J 《Proteins》2009,74(2):449-460
In this article, we present a new method termed CatFam (Catalytic Families) to automatically infer the functions of catalytic proteins, which account for 20-40% of all proteins in living organisms and play a critical role in a variety of biological processes. CatFam is a sequence-based method that generates sequence profiles to represent and infer protein catalytic functions. CatFam generates profiles through a stepwise procedure that carefully controls profile quality and employs nonenzymes as negative samples to establish profile-specific thresholds associated with a predefined nominal false-positive rate (FPR) of predictions. The adjustable FPR allows for fine precision control of each profile and enables the generation of profile databases that meet different needs: function annotation with high precision and hypothesis generation with moderate precision but better recall. Multiple tests of CatFam databases (generated with distinct nominal FPRs) against enzyme and nonenzyme datasets show that the method's predictions have consistently high precision and recall. For example, a 1% FPR database predicts protein catalytic functions for a dataset of enzymes and nonenzymes with 98.6% precision and 95.0% recall. Comparisons of CatFam databases against other established profile-based methods for the functional annotation of 13 bacterial genomes indicate that CatFam consistently achieves higher precision and (in most cases) higher recall, and that (on average) CatFam provides 21.9% additional catalytic functions not inferred by the other similarly reliable methods. These results strongly suggest that the proposed method provides a valuable contribution to the automated prediction of protein catalytic functions. The CatFam databases and the database search program are freely available at http://www.bhsai.org/downloads/catfam.tar.gz.  相似文献   

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