Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes.
Toll-like receptors (TLRs) and members of their signalling pathway play an important role in the initiation of the innate immune response to a wide variety of pathogens1,2,3. The adaptor protein TIRAP mediates downstream signalling of 5,6. We report a case-control genetic association study of 6106 individuals from Gambia, Kenya, United Kingdom, and Vietnam, with invasive pneumococcal disease, bacteraemia, malaria and tuberculosis. Thirty-three SNPs were genotyped, including TIRAP S180L. Heterozygous carriage of this variant was found to associate independently with all four infectious diseases in the different study populations (P=0.003, OR=0.59, 95%CI 0.42-0.83 for IPD; P=0.003, OR=0.40, 95%CI 0.21-0.77 for bacteraemia; P=0.002, OR=0.47, 95%CI 0.28-0.76 for malaria; P=0.008, OR=0.23 95%CI 0.07-0.73 for tuberculosis). Substantial support for a protective effect of S180L heterozygosity against infectious diseases was observed when the study groups were combined (N=6106, OverallCorrespondence should be addressed on genetics to AVSH (adrian.hill@well.ox.ac.uk) In the UK population, heterozygosity at TIRAP S180L was associated with protection from invasive pneumococcal disease (3×2 χ 2 =8.72, P=0.013, Table 1). An excess of mutant homozygotes amongst IPD cases (Table 1) was also observed in this UK population. TIRAP S180L was then examined in a separate group of UK individuals with thoracic empyema and a second control group. Although no association was observed between genotype and susceptibility to thoracic empyema overall (n=584, 3×2 χ 2 =0.63, P=0.73), analysis of the small subgroup of individuals with pneumococcal empyema revealed a non-significant trend towards association (3×2 χ 2 =5.05, P=0.080; Table 1). Interestingly, an excess of mutant homozygotes was again observed amongst this second group of IPD cases (Table 1).We then studied TIRAP S180L in a second population with invasive bacterial disease, comprising Kenyan children with well-defined bacteraemia. Although the mutant allele was found to be less common in the Kenyan population than in UK individuals, the same pattern of association was observed. The TIRAP S180L heterozygotes were significantly more common amongst community controls (5.9%), compared to individuals with bacteraemia (2.4%) (2×2 χ 2 =9.05, P=0.003; Table 1). The heterozygote protective effect of the S180L locus was also significant within the subgroup of 164 Kenyan children with pneumococcal bacteraemia (F exact =0.024, Table 1), thus replicating the findings in the UK studies.In the Gambian malaria case-control study, TIRAP S180L heterozygosity demonstrated a significant protective effect against both general malaria (Wald=8.35, P=0.004, Table 1) and severe malaria (Wald=8.706, P=0.003, Table 1). This result was replicated in a second malaria case-control study, this time in a Vietnamese population whose design included only cases of severe malaria: TIRAP S180L heterozygotes were again found to be more prevalent Finally, the possible effect of the TIRAP S180L polymorphism on ...
Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.
Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.immunity | antigen recognition | X-ray crystallography E xposure to a novel antigen, whether by infection or vaccination, induces an initial naive B-cell response. Cells bearing B-cell receptors (BCRs) that bind the antigen in question, even with relatively low affinity, proliferate selectively. In the continued presence of antigen, additional proliferation, accompanied by somatic hypermutation of the rearranged Ig heavy-and light-chain genes, leads to selection of cells with BCRs (and secreted antibodies) that bind more tightly to antigen than their precursors-a process known as affinity maturation (1-3). Recent methodological advances make it possible to study the history of this process in a given subject by isolating a number of individual B cells at a suitable time point after vaccination or infection and cloning their recombined heavy-and light-chain variable regions (4-6). If a subset of the variable regions thus identified derives from the same progenitor, one can infer the clonal lineage that gave rise to the observed genes, including the unmutated common ancestor (UCA) and the other unobserved intermediates at the interior nodes of the clonal tree with tips that are the genes of the mature antibodies (Fig. 1A). Structural and biochemical changes that occur during affinity maturation can be analyzed, and the mechanism of affinity enhancement elucidated.The influenza B-cell clonal lineage shown in Fig. 1A derives from plasmablasts sorted from a sample taken from an adult subject 1 wk after administration of the 2007 trivalent inactivated influenza virus vaccine. It includes just three mature B-cell clones. We have shown that one member of this lineage (CH65) bears a heavy-chain complementary determining region 3 (CDR H3) loop that inserts into the HA receptor-binding pocket, mimics the influenza virus receptor sialic acid, and has unusual breadth of neutralizing capacity (31 of 36 H1 strains tested) (7). We have now extended the structural and functional analysis to the entire lineage. By determining the structure and binding properties of the UCA and intermediate 2...
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