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1.
A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible “lid” anchored in the active site. However, the mechanistic details of the “lid” displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its “lid” mutant and proposed a role of the “lid” as a protector of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation. 相似文献
2.
Orna Almog Itai Benhar George Vasmatzis Maria Tordova Byungkook Lee Ira Pastan Gary L. Gilliland 《Proteins》1998,31(2):128-138
A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023–1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1-Å resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Å wide and 17-Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc–Gal–Nag–Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781–3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128–138, 1998. Published 1998 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America. 相似文献
3.
Pizarro JC Chitarra V Verger D Holm I Pêtres S Dartevelle S Nato F Longacre S Bentley GA 《Journal of molecular biology》2003,328(5):1091-1103
Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing. 相似文献
4.
Strelkov SV Schumacher J Burkhard P Aebi U Herrmann H 《Journal of molecular biology》2004,343(4):1067-1080
Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation. 相似文献
5.
Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ from Vibrio species interact directly with one another. Moreover, the 2.3 Å resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, “variable pilin segment” seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus. 相似文献
6.
A multi-copper protein with two cupredoxin-like domains was identified from our in-house metagenomic database. The recombinant protein, mgLAC, contained four copper ions/subunits, oxidized various phenolic and non-phenolic substrates, and had spectroscopic properties similar to common laccases. X-ray structure analysis revealed a homotrimeric architecture for this enzyme, which resembles nitrite reductase (NIR). However, a difference in copper coordination was found at the domain interface. mgLAC contains a T2/T3 tri-nuclear copper cluster at this site, whereas a mononuclear T2 copper occupies this position in NIR. The trimer is thus an essential part of the architecture of two-domain multi-copper proteins, and mgLAC may be an evolutionary precursor of NIR. 相似文献
7.
The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin-ligand binding has been extensively studied because of the potential for the development of anti-adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N-terminus of the β subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A-domain, thereby potentially placing a previously characterised module at the centre of a key functional region. 相似文献
8.
An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and theras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the subunit of F1. Several potentially important residues on are noted that have not yet been studied by mutagenesis or affinity labeling. 相似文献
9.
We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter-chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14–41 of PI. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22–37 (numbered with respect to P1) form pairs of α-helices, with no evidence for any other secondary structure. © 1994 Wiley-Liss, Inc. 相似文献
10.
Runying Yang 《生物化学与生物物理学报:生物膜》2008,1778(2):454-465
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the β-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 °C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the β-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport. 相似文献