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1.
Miller DJ Shuvalova L Evdokimova E Savchenko A Yakunin AF Anderson WF 《Protein science : a publication of the Protein Society》2007,16(7):1338-1348
Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis-p-nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 A resolution. YfcE has a beta-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique. 相似文献
2.
Zinc is one of the biologically most abundant and important metal elements, present in a plethora of enzymes from a broad array of species of all phyla. In this study we report a thorough analysis of the geometrical properties of Zinc coordination spheres performed on a dataset of 994 high quality protein crystal structures from the Protein Data Bank, and complemented with Quantum mechanical calculations at the DFT level of theory (B3LYP/SDD) on mononuclear model systems. The results allowed us to draw interesting conclusions on the structural characteristics of Zn centres and to evaluate the importance of such effects as the resolution of X-ray crystallographic structures, the enzyme class in which the Zn centre is included, and the identity of the ligands at the Zn coordination sphere. Altogether, the set of results obtained provides useful data for the enhancement of the atomic models normally applied to the theoretical and computational study of zinc enzymes at the quantum mechanical level (in particular enzymatic mechanisms), and for the development of molecular mechanical parameters for the treatment of zinc coordination spheres with molecular mechanics or molecular dynamics in studies with the full enzyme. 相似文献
3.
The rapid accumulation of gene sequences, many of which are hypothetical proteins with unknown function, has stimulated the development of accurate computational tools for protein function prediction with evolution/structure‐based approaches showing considerable promise. In this article, we present FINDSITE‐metal, a new threading‐based method designed specifically to detect metal‐binding sites in modeled protein structures. Comprehensive benchmarks using different quality protein structures show that weakly homologous protein models provide sufficient structural information for quite accurate annotation by FINDSITE‐metal. Combining structure/evolutionary information with machine learning results in highly accurate metal‐binding annotations; for protein models constructed by TASSER, whose average Cα RMSD from the native structure is 8.9 Å, 59.5% (71.9%) of the best of top five predicted metal locations are within 4 Å (8 Å) from a bound metal in the crystal structure. For most of the targets, multiple metal‐binding sites are detected with the best predicted binding site at rank 1 and within the top two ranks in 65.6% and 83.1% of the cases, respectively. Furthermore, for iron, copper, zinc, calcium, and magnesium ions, the binding metal can be predicted with high, typically 70% to 90%, accuracy. FINDSITE‐metal also provides a set of confidence indexes that help assess the reliability of predictions. Finally, we describe the proteome‐wide application of FINDSITE‐metal that quantifies the metal‐binding complement of the human proteome. FINDSITE‐metal is freely available to the academic community at http://cssb.biology.gatech.edu/findsite‐metal/ . Proteins 2011. © 2010 Wiley‐Liss, Inc. 相似文献
4.
Hoang Nguyen Jesse Kleingardner 《Protein science : a publication of the Protein Society》2021,30(6):1247
Metal cofactors within proteins perform a versatile set of essential cellular functions. In order to take advantage of the diverse functionality of metalloproteins, researchers have been working to design or modify metal binding sites in proteins to rationally tune the function or activity of the metal cofactor. This study has performed an analysis on the backbone atom geometries of metal‐binding amino acids among 10 different metal binding sites within the entire protein data bank. A set of 13 geometric parameters (features) was identified that is capable of predicting the presence of a metal cofactor in the protein structure with overall accuracies of up to 97% given only the relative positions of their backbone atoms. The decision tree machine‐learning algorithm used can quickly analyze an entire protein structure for the presence of sets of primary metal coordination spheres upon mutagenesis, independent of their original amino acid identities. The methodology was designed for application in the field of metalloprotein engineering. A cluster analysis using the data set was also performed and demonstrated that the features chosen are useful for identifying clusters of structurally similar metal‐binding sites. 相似文献
5.
Ajitha S. Cristie‐David E. Neil G. Marsh 《Protein science : a publication of the Protein Society》2019,28(9):1620-1629
Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni2+, Co2+, Cu2+, and Zn2+ ions were evaluated, with Ni2+ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions. 相似文献
6.
Maria Elena Compostella Paola Berto Francesca Vallese Giuseppe Zanotti 《Acta Crystallographica. Section F, Structural Biology Communications》2015,71(8):1005-1011
The crystal structure of α‐carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacterium has corrected for the mutation of an essential amino acid important for catalysis using a negative ion from the medium and how it localizes close to the inner membrane in the periplasm. Since carbonic anhydrase is essential for the bacterial colonization of the host, it is a potential target for antibiotic drugs. The definition of the shape of the active‐site entrance and cavity constitutes a basis for the design of specific inhibitors. 相似文献
7.
Marco Barba Anatoli P. Sobolev Cristina Romeo M. Eugenia Schininà Donatella Pietraforte Luisa Mannina Giovanni Musci Fabio Polticelli 《Protein science : a publication of the Protein Society》2009,18(3):559-568
Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post‐translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well‐defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox‐active (macro)molecules was tested by engineering a copper‐binding site on two different variants of the natural peptide Contryphan‐Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg–Cupryphan, bind Cu2+ ions with a 1:1 stoichiometry and a Kd in the 100 nM range. Other divalent metals (e.g., Zn2+ and Mg2+) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins. 相似文献
8.
Bryan PN 《Biotechnology advances》1987,5(2):221-224
The techniques of protein engineering are proving to be a revolutionary experimental tool for understanding protein structure-function relationships. Even at this early stage, proteins of improved characteristics for specific industrial and therapeutic uses have already been produced. Tailoring enzymatic properties for non-physiological substrate conditions, altering pH optima, changing substrate specificity, and improving stability have already been demonstrated to be feasible. Nevertheless, the ability to make useful proteins which radically differ from a natural structure or designing altogether new structures exceeds present understanding. 相似文献
9.
Craig A. Behnke Isolde Le Trong Jeff W. Godden Ethan A. Merritt David C. Teller Jürgen Bajorath Ronald E. Stenkamp 《Acta Crystallographica. Section D, Structural Biology》2010,66(5):616-627
Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X‐ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide‐containing inhibitor complex has been carried out and the structure was refined at 0.9 Å resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active‐site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 Å) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 Å in some zinc–protein and zinc–ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron‐density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X‐ray structures, even if atomic resolution data are available. 相似文献
10.
Lucia Banci Ivano Bertini Mauro Andrea Cremonini Giovanni Gori-Savellini Claudio Luchinat Kurt Wüthrich Peter Güntert 《Journal of biomolecular NMR》1998,12(4):553-557
The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints. 相似文献
11.
NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E. coli glutaredoxin and functionally related proteins. 
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下载免费PDF全文 T. H. Xia J. H. Bushweller P. Sodano M. Billeter O. Bjrnberg A. Holmgren K. Wüthrich 《Protein science : a publication of the Protein Society》1992,1(3):310-321
The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible. 相似文献
12.
Chu Wang Robert Vernon Oliver Lange Michael Tyka David Baker 《Protein science : a publication of the Protein Society》2010,19(3):494-506
Metal ions play an essential role in stabilizing protein structures and contributing to protein function. Ions such as zinc have well‐defined coordination geometries, but it has not been easy to take advantage of this knowledge in protein structure prediction efforts. Here, we present a computational method to predict structures of zinc‐binding proteins given knowledge of the positions of zinc‐coordinating residues in the amino acid sequence. The method takes advantage of the “atom‐tree” representation of molecular systems and modular architecture of the Rosetta3 software suite to incorporate explicit metal ion coordination geometry into previously developed de novo prediction and loop modeling protocols. Zinc cofactors are tethered to their interacting residues based on coordination geometries observed in natural zinc‐binding proteins. The incorporation of explicit zinc atoms and their coordination geometry in both de novo structure prediction and loop modeling significantly improves sampling near the native conformation. The method can be readily extended to predict protein structures bound to other metal and/or small chemical cofactors with well‐defined coordination or ligation geometry. 相似文献
13.
Tynan-Connolly BM Nielsen JE 《Protein science : a publication of the Protein Society》2007,16(2):239-249
The ability to re-engineer enzymatic pH-activity profiles is of importance for industrial applications of enzymes. We theoretically explore the feasibility of re-engineering enzymatic pH-activity profiles by changing active site pK(a) values using point mutations. We calculate the maximum achievable DeltapK(a) values for 141 target titratable groups in seven enzymes by introducing conservative net-charge altering point mutations. We examine the importance of the number of mutations introduced, their distance from the target titratable group, and the characteristics of the target group itself. The results show that multiple mutations at 10A can change pK(a) values up to two units, but that the introduction of a requirement to keep other pK(a) values constant reduces the magnitude of the achievable DeltapK(a). The algorithm presented shows a good correlation with existing experimental data and is available for download and via a web server at http://enzyme.ucd.ie/pKD. 相似文献
14.
N. J. Hess G. W. Buchko S. D. Conradson F. J. Espinosa S. Ni B. D. Thrall M. A. Kennedy 《Protein science : a publication of the Protein Society》1998,7(9):1970-1975
The ubiquitous, multi-enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219). The zinc-associated motif is essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (CoXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33+/-0.02 A. 相似文献
15.
A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility. 相似文献
16.
Structure comparison is widely used to quantify protein relationships. Although there are several approaches to calculate structural similarity, specifying significance thresholds for similarity metrics is difficult due to the inherent likeness of common secondary structure elements. In this study, metal co‐factor location is used to assess the biological relevance of structural alignments. The distance between the centroids of bound co‐factors adds a chemical and function‐relevant constraint to the structural superimposition of two proteins. This additional dimension can be used to define cut‐off values for discriminating valid and spurious alignments in large alignment sets. The hypothesis underlying our approach is that metal coordination sites constrain structural evolution, thus revealing functional relationships between distantly related proteins. A comparison of three related nitrogenases shows the sequence and fold constraints imposed on the protein structures up to 18 Å away from the centers of their bound metal clusters. Proteins 2014; 82:648–656. © 2013 Wiley Periodicals, Inc. 相似文献
17.
Yu An Genpei Li Binggen Ru 《Acta Crystallographica. Section D, Structural Biology》1999,55(6):1242-1243
Metallothionein II (Mw = 6.8 kDa), induced by cadmium and purified from rabbit liver, has been crystallized in space group P6222 or P6422. The unit-cell parameters were a = b = 113.4, c = 219.1 Å, α = β = 90.0, γ = 120.0° when crystallized from sodium citrate buffer and a = b = 113.4, c = 219.6 Å, α = β = 90.0, γ = 120.0° when crystallized from Tris–HCl buffer. There are 12 molecules per asymmetric unit and the solvent content is about 57%. 相似文献
18.
Mochalkin I Knafels JD Lightle S 《Protein science : a publication of the Protein Society》2008,17(3):450-457
The cell wall in Gram-negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is an essential zinc-dependant enzyme that catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)glucosamine and acetate in the biosynthesis of lipid A, and for this reason, LpxC is an attractive target for antibacterial drug discovery. Here we disclose a 1.9 A resolution crystal structure of LpxC from Pseudomonas aeruginosa (paLpxC) in a complex with the potent BB-78485 inhibitor. To our knowledge, this is the first crystal structure of LpxC with a small-molecule inhibitor that shows antibacterial activity against a wide range of Gram-negative pathogens. Accordingly, this structure can provide important information for lead optimization and rational design of the effective small-molecule LpxC inhibitors for successful treatment of Gram-negative infections. 相似文献
19.
Svetlana Pakhomova William E. Boeglin David B. Neau Sue G. Bartlett Alan R. Brash Marcia E. Newcomer 《Protein science : a publication of the Protein Society》2019,28(5):920-927
The regio‐ and stereo‐specific oxygenation of polyunsaturated fatty acids is catalyzed by lipoxygenases (LOX); both Fe and Mn forms of the enzyme have been described. Structural elements of the Fe and Mn coordination spheres and the helical catalytic domain in which the metal center resides are highly conserved. However, animal, plant, and microbial LOX each have distinct features. We report five crystal structures of a LOX from the fungal plant pathogen Fusarium graminearum. This LOX displays a novel amino terminal extension that provides a wrapping domain for dimerization. Moreover, this extension appears to interfere with the iron coordination sphere, as the typical LOX configuration is not observed at the catalytic metal when the enzyme is dimeric. Instead novel tetra‐, penta‐, and hexa‐coordinate Fe2+ ligations are apparent. In contrast, a monomeric structure indicates that with repositioning of the amino terminal segment, the enzyme can assume a productive conformation with the canonical Fe2+ coordination sphere. 相似文献
20.
Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron‐uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at “catalytic” NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific “iron‐uptake‐competent” conformations of apoRd on the NP surface. This bio‐mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor‐triggered refolding, particularly when over‐expressed under limited cofactor accessibility. Proteins 2014; 82:3154–3162. © 2014 Wiley Periodicals, Inc. 相似文献