Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.
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ABSTRACT: A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.
Project description:Limited or regulatory proteolysis plays a critical role in many important biological pathways like blood coagulation, cell proliferation, and apoptosis. A better understanding of mechanisms that control this process is required for discovering new proteolytic events and for developing inhibitors with potential therapeutic value. Two features that determine the susceptibility of peptide bonds to proteolysis are the sequence in the vicinity of the scissile bond and the structural context in which the bond is displayed. In this study, we assessed statistical significance and predictive power of individual structural descriptors and combination thereof for the identification of cleavage sites. The analysis was performed on a data set of >200 proteolytic events documented in CutDB for a variety of mammalian regulatory proteases and their physiological substrates with known 3D structures. The results confirmed the significance and provided a ranking within three main categories of structural features: exposure > flexibility > local interactions. Among secondary structure elements, the largest frequency of proteolytic cleavage was confirmed for loops and lower but significant frequency for helices. Limited proteolysis has lower albeit appreciable frequency of occurrence in certain types of ?-strands, which is in contrast with some previous reports. Descriptors deduced directly from the amino acid sequence displayed only marginal predictive capabilities. Homology-based structural models showed a predictive performance comparable to protein substrates with experimentally established structures. Overall, this study provided a foundation for accurate automated prediction of segments of protein structure susceptible to proteolytic processing and, potentially, other post-translational modifications.
Project description:Elucidation of the structure of PrP(Sc) continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP(Sc)). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP(Sc) from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP(Sc) samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP(Sc). These results reinforce the hypothesis that the structure of PrP(Sc) consists of a series of highly PK-resistant ?-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP(Sc) is highly resistant to PK and therefore perhaps also contains ?-sheet secondary structure.
Project description:Planctomycetes, Verrucomicrobia and Chlamydia are prokaryotic phyla, sometimes grouped together as the PVC superphylum of eubacteria. Some PVC species possess interesting attributes, in particular, internal membranes that superficially resemble eukaryotic endomembranes. Some biologists now claim that PVC bacteria are nucleus-bearing prokaryotes and are considered evolutionary intermediates in the transition from prokaryote to eukaryote. PVC prokaryotes do not possess a nucleus and are not intermediates in the prokaryote-to-eukaryote transition. Here we summarise the evidence that shows why all of the PVC traits that are currently cited as evidence for aspiring eukaryoticity are either analogous (the result of convergent evolution), not homologous, to eukaryotic traits; or else they are the result of horizontal gene transfers.
Project description:The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors.
Project description:The primary structure of the second component of human complement (C2) was determined by cDNA cloning and sequence analysis. C2 has 39% identity with the functionally analogous protein Factor B. The C-terminal half of C2a is homologous to the catalytic domains of other serine proteinases. C2b contains three direct repeats of approx. 60 amino acid residues. They are homologous to repeats in Factor B, C4b-binding protein and Factor H, suggesting a functional significance of the repeat in C4b and C3b binding. The repeats are also found in the non-complement proteins beta 2-glycoprotein I and interleukin-2 receptor, and this repeat family may be widespread.
Project description:Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.
Project description:Very solid evidence suggests that the core of full length PrPSc is a 4-rung ?-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the ?-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of ?-strands, helping us to predict the threading of the ?-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.
Project description:The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.
Project description:Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.
Project description:Using a microtitre plate assay, direct binding between complement factors I and H was demonstrated, and ligand blotting indicated that factor H interacts with the heavy chain of factor I. Similarly, direct C3(NH3)-factor I and C3(NH3)-factor H binding was characterized [where C3(NH3) is a form of C3 that is cleaved by factor I in the presence of factor H]. Both factor H and factor I interacted with both chains of C3(NH3) in ligand blotting. Binding reactions between all three pairs of components were highly dependent on ionic strength, and showed similar pH optima. Binding assays with all three components present led to the following conclusions. (a) Binding sites for C3(NH3) and factor I on factor H do not overlap, and binding of factor I and C3(NH3) to soluble factor H promotes the weak factor I-C3(NH3) interaction. (b) Anomalies arise with immobilized factor H, which may be artefactual or may reflect the physiological situation. (c) Similarly, binding sites on factor I for C3(NH3) and for factor H do not overlap, and binding of factor H and C3(NH3) to factor I promotes direct factor H-C3(NH3) interactions. Based on these results, a model of the interactions between factor H, factor I and C3(NH3) leading to the processing of C3(NH3) is proposed.