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1.
Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease 总被引:2,自引:0,他引:2
The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild-type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central beta-sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin. 相似文献
2.
Serpins are a family of structurally homologous proteins having metastable native structures. As a result, a serpin variant destabilized by mutation(s) has a tendency to undergo conformational changes leading to inactive forms, e.g., the latent form and polymer. Serpin polymers are involved in a number of conformational diseases. Although several models for polymer structure have been proposed, the actual structure remains unknown. Here, we provide a comprehensive list of serpins, both free and in complexes, deposited in the Protein Data Bank. Our discussion focuses on structures that potentially can contribute to a better understanding of polymer structure. 相似文献
3.
Peter Gettins Philip A. Patston Marc Schapira 《BioEssays : news and reviews in molecular, cellular and developmental biology》1993,15(7):461-467
Serpins are members of a family of structurally related protein inhibitors of serine proteinases, with molecular masses between 40 and 100kDa. In contrast to other, simpler, proteinase inhibitors, they may interact with proteinases as inhibitors, as substrates, or as both. They undergo conformational interconversions upon complex formation with proteinase, upon binding of some members to heparin, upon proteolytic cleavage at the reactive center, and under mild denaturing conditions. These conformational changes appear to be critical in determining the properties of the serpin. The structures and stabilities of these various forms may differ significantly. Although the detailed structural changes required for inhibition of proteinase have yet to be worked out, it is clear that the serpin does undergo a major conformational change. This is in contrast to other, simpler, families of protein inhibitors of serine proteinases, which bind in a substrate-like or product-like manner. Proteolytic cleavage of the serpin can result in a much more stable protein with new biological properties such as chemo-attractant behaviour. These structural transformations in serpins provide opportunities for regulation of the activity and properties of the inhibitor and are likely be important in vivo, where serpins are involved in blood coagulation, fibrinolysis, complement activation and inflammation. 相似文献
4.
Beinrohr L Harmat V Dobó J Lörincz Z Gál P Závodszky P 《The Journal of biological chemistry》2007,282(29):21100-21109
C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. 相似文献
5.
Irving JA Cabrita LD Rossjohn J Pike RN Bottomley SP Whisstock JC 《Structure (London, England : 1993)》2003,11(4):387-397
Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C. The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment. 相似文献
6.
Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen. 相似文献
7.
S J Harrop L Jankova M Coles D Jardine J S Whittaker A R Gould A Meister G C King B C Mabbutt P M Curmi 《Structure (London, England : 1993)》1999,7(1):43-54
BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease. 相似文献
8.
Pearce MC Cabrita LD Rubin H Gore MG Bottomley SP 《Biochemical and biophysical research communications》2004,324(2):729-735
The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed. 相似文献
9.
Lee CC Ko TP Chou CC Yoshimura M Doong SR Wang MY Wang AH 《Journal of structural biology》2006,155(1):74-86
The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes. 相似文献
10.
Gorlatova NV Elokdah H Fan K Crandall DL Lawrence DA 《The Journal of biological chemistry》2003,278(18):16329-16335
The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is approximately 100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition. 相似文献
11.
Nar H Bauer M Stassen JM Lang D Gils A Declerck PJ 《Journal of molecular biology》2000,297(3):683-695
The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A.The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered.The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A.The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin. 相似文献
12.
A K Sharma M Paramasivam A Srinivasan M P Yadav T P Singh 《Journal of molecular biology》1999,289(2):303-317
Lactoferrin is a monomeric glycoprotein with a molecular mass of approximately 80 kDa. The three-dimensional structure of mare diferric lactoferrin (mlf) has been determined at 2.6 A resolution. The protein crystallizes in the space group P 212121with a=85.2 A, b=99.5 A, c=103.1 A with a solvent content of 55 % (v/v). The structure was solved by the molecular replacement method using human diferric lactoferrin as the model. The structure has been refined using XPLOR to a final R -factor of 0.194 for all data in the 15-2.6 A resolution range. The amino acid sequence of mlf was determined using a cDNA method. The final refined model comprises 5281 protein atoms, 2 Fe3+, 2 CO32-and 112 water molecules. The overall folding of mlf is similar to that of other proteins of the transferrin family. The protein folds into two globular lobes, N and C. The lobes are further divided into two domains, N1 and N2, and C1 and C2. The iron-binding cleft is situated between the domains in each lobe. The N lobe appears to be well ordered and is more stable than the C lobe in mlf unlike in other lactoferrins, where the C lobe is the more stable. The opening of the binding cleft in the N lobe of mlf is narrower than those in other proteins of the transferrin family. This is very unusual and is found only in mare lactoferrin. Apart from certain hydrophobic interactions at the mouth of the cleft, one salt-bridge (Lys301 . . . . . . . . Glu216) crosses between the two walls of the cleft. The two lobes are connected covalently by a three-turn alpha-helix involving residues 334-344. The N lobe displays a highly ordered structure with appreciably low temperature factors. The iron coordination is more symmetrical in the N lobe than in the C lobe. There are only 16 intermolecular hydrogen bonds in the structure of mlf. 相似文献
13.
Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers. 相似文献
14.
Matthias Kronen Helmar G?rls Hoai-Huong Nguyen Siegmund Reissmann Martin Bohl Jürgen Sühnel Udo Gr?fe 《Journal of peptide science》2003,9(11-12):729-744
Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A. 相似文献
15.
Bush DJ Kirillova O Clark SA Davulcu O Fabiola F Xie Q Somasundaram T Ellington WR Chapman MS 《The Journal of biological chemistry》2011,286(11):9338-9350
Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. 相似文献
16.
The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding 总被引:19,自引:0,他引:19
The three-dimensional structure of tumor necrosis factor (TNF-alpha), a protein hormone secreted by macrophages, has been determined at 2.6 A resolution by x-ray crystallography. Phases were determined by multiple isomorphous replacement using data collected from five heavy atom derivatives. The multiple isomorphous replacement phases were further improved by real space symmetry averaging, exploiting the noncrystallographic 3-fold symmetry of the TNF-alpha trimer. An atomic model corresponding to the known amino acid sequence of TNF-alpha was readily built into the electron density map calculated with these improved phases. The 17,350-dalton monomer forms an elongated, antiparallel beta-pleated sheet sandwich with a "jelly-roll" topology. Three monomers associate intimately about a 3-fold axis of symmetry to form a compact bell-shaped trimer. Examination of the model and comparison to known protein structures reveals striking structural homology to several viral coat proteins, particularly satellite tobacco necrosis virus. Locations of residues conserved between TNF-alpha and lymphotoxin (TNF-beta, a related cytokine known to bind to the same receptors as TNF-alpha) suggest that lymphotoxin, like TNF-alpha, binds to the receptor as a trimer and that the general site of interaction with the receptor is at the "base" of the trimer. 相似文献
17.
Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution.
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The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication. 相似文献
18.
19.
We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests (as did earlier probe experiments) that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5-0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface. 相似文献
20.
Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications. 相似文献