Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.     

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.     

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.     

Energetic evaluation of binding modes in the C3d and Factor H (CCP 19-20) complex

As a part of innate immunity, the complement system relies on activation of the alternative pathway (AP). While feed-forward amplification generates an immune response towards foreign surfaces, the process requires regulation to prevent an immune response on the surface of host cells. Factor H (FH) is a complement protein secreted by native cells to negatively regulate the AP. In terms of structure, FH is composed of 20 complement-control protein (CCP) modules that are structurally homologous but vary in composition and function. Mutations in these CCPs have been linked to states of autoimmunity. In particular, several mutations in CCP 19-20 are correlated to atypical hemolytic uremic syndrome (aHUS). From crystallographic structures there are three putative binding sites of CCP 19-20 on C3d. Since there has been some controversy over the primary mode of binding from experimental studies, we approach characterization of binding using computational methods. Specifically, we compare each binding mode in terms of electrostatic character, structural stability, dissociative and associative properties, and predicted free energy of binding. After a detailed investigation, we found two of the three binding sites to be similarly stable while varying in the number of contacts to C3d and in the energetic barrier to complex dissociation. These sites are likely physiologically relevant and may facilitate multivalent binding of FH CCP 19-20 to C3b and either C3d or host glycosaminoglycans. We propose thermodynamically stable binding with modules 19 and 20, the latter driven by electrostatics, acting synergistically to increase the apparent affinity of FH for host surfaces.     

IGFBP7基因的结构与功能的研究进展

胰岛素样生长因子结合蛋白7（IGFBP7）是IGFBPs超家族的新成员,结构上除具有与IGFBPs相似的保守N端结构域外,还有特异的Kazal型丝氨酸蛋白酶抑制结构域和免疫球蛋白样C2结构域。除与IGFs结合发挥作用外,还能独立调控细胞凋亡、增殖和迁移等。而至今尚无对水生无脊椎动物IGFBP7的研究报道,结合本实验室的研究综述了目前IGFBP7基因结构和功能上的研究进展,并对今后的研究工作进行了展望。     

Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C_16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C_18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C_16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes.     

Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H

Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.     

NC4 Domain of cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.     

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation

Insulin-like growth factor binding protein (IGFBP)-6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP-6 effects on osteoblast differentiation. During MC3T3-E1 osteoblast differentiation, we found that IGFBP-6 protein was down-regulated. Overexpression of IGFBP-6 in MC3T3-E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP-6 in the culture media was not required for any of these effects suggesting that IGFBP-6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two-hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP-6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP-1) was identified as a high affinity IGFBP-6 binding partner. Physical interaction between IGFBP-6 and LMP-1 was confirmed by co-immunoprecipitation. Fluorescent protein fusion constructs for LMP-1 and IGFBP-6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP-1-GFP and IGFBP-6-RFP resulted in overlapping subcellular localization of LMP-1 and IGFBP-6. To determine if there was a functional association of IGFBP-6 and LMP-1 as well as a physical association, we studied the effect of IGFBP-6, LMP-1 and their combination on type I procollagen promoter activity. LMP-1 increased promoter activity while IGFBP-6 reduced promoter activity, and coexpression of LMP-1 with IGFBP-6 abrogated IGFBP-6 suppression. These studies provide evidence that overexpression of IGFBP-6 suppresses human and murine osteoblast differentiation, that IGFBP-6 and LMP-1 physically interact, and supports the conclusion that this interaction may be functionally relevant.     

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

Using collections of structural models to predict changes of binding affinity caused by mutations in protein–protein interactions

Protein–protein interactions (PPIs) in all the molecular aspects that take place both inside and outside cells. However, determining experimentally the structure and affinity of PPIs is expensive and time consuming. Therefore, the development of computational tools, as a complement to experimental methods, is fundamental. Here, we present a computational suite: MODPIN, to model and predict the changes of binding affinity of PPIs. In this approach we use homology modeling to derive the structures of PPIs and score them using state‐of‐the‐art scoring functions. We explore the conformational space of PPIs by generating not a single structural model but a collection of structural models with different conformations based on several templates. We apply the approach to predict the changes in free energy upon mutations and splicing variants of large datasets of PPIs to statistically quantify the quality and accuracy of the predictions. As an example, we use MODPIN to study the effect of mutations in the interaction between colicin endonuclease 9 and colicin endonuclease 2 immune protein from Escherichia coli. Finally, we have compared our results with other state‐of‐art methods.     

Adaptation of avian influenza A (H6N1) virus from avian to human receptor‐binding preference

The receptor‐binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor‐binding properties of Taiwan‐isolated H6 HAs from 1972 to 2013. We propose that the receptor‐binding properties of Taiwan‐isolated H6 HAs have undergone three major stages: initially avian receptor‐binding preference, secondarily obtaining human receptor‐binding capacity, and recently human receptor‐binding preference, which has been confirmed by receptor‐binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor‐binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor‐binding site of HA determines the receptor‐binding preference change. We conclude that the human‐infecting H6N1 evolved into a human receptor preference.     

Multiple binding of type 3 streptococcal M protein to human fibrinogen, albumin and fibronectin

Abstract M proteins are major virulence factors of group A streptococci which enable the bacteria to resist phagocytic attack. Their binding capacity for different plasma proteins seems to be one reason for the antiphagocytic activity of M protein. In the present study we demonstrate that M3 protein, isolated from the streptococcal culture supernatant of strain 4/55, and the recombinant form (rM3), purified from an E. coli lysate after cloning in phage γ-EMBL3, show a multiple binding to fibrinogen, albumin and fibronectin in Western blot and dot binding assays. Binding of M3 protein to the multifunctional extracellular matrix and plasma protein fibronectin may not only influence phagocytosis but may also contribute to the adherence of these bacteria to endothelial and epithelial cells.     

Classification and annotation of the relationship between protein structural change and ligand binding

The causal relationship between protein structural change and ligand binding was classified and annotated for 839 nonredundant pairs of crystal structures in the Protein Data Bank—one with and the other without a bound low-molecular-weight ligand molecule. Protein structural changes were first classified into either domain or local motions depending on the size of the moving protein segments. Whether the protein motion was coupled with ligand binding was then evaluated based on the location of the ligand binding site and by application of the linear response theory of protein structural change. Protein motions coupled with ligand binding were further classified into either closure or opening motions. This classification revealed the following: (i) domain motions coupled with ligand binding are dominated by closure motions, which can be described by the linear response theory; (ii) local motions frequently accompany order-disorder or α-helix-coil conformational transitions; and (iii) transferase activity (Enzyme Commission   number 2) is the predominant function among coupled domain closure motions. This could be explained by the closure motion acting to insulate the reaction site of these enzymes from environmental water.