A new map of glycosaminoglycan and C3b binding sites on factor H

Human complement factor H, consisting of 20 complement control protein (CCP) modules, is an abundant plasma glycoprotein. It prevents C3b amplification on self surfaces bearing certain polyanionic carbohydrates, while complement activation progresses on most other, mainly foreign, surfaces. Herein, locations of binding sites for polyanions and C3b are reexamined rigorously by overexpressing factor H segments, structural validation, and binding assays. As anticipated, constructs corresponding to CCPs 7-8 and 19-20 bind well in heparin-affinity chromatography. However, CCPs 8-9, previously reported to bind glycosaminoglycans, bind neither to heparin resin nor to heparin fragments in gel-mobility shift assays. Introduction of nonnative residues N-terminal to a construct containing CCPs 8-9, identical to those in proteins used in the previous report, converted this module pair to an artificially heparin-binding one. The module pair CCPs 12-13 does not bind heparin appreciably, notwithstanding previous suggestions to the contrary. We further checked CCPs 10-12, 11-14, 13-15, 10-15, and 8-15 for ability to bind heparin but found very low affinity or none. As expected, constructs corresponding to CCPs 1-4 and 19-20 bind C3b amine coupled to a CM5 chip (K(d)s of 14 and 3.5 microM, respectively) or a C1 chip (K(d)s of 10 and 4.5 microM, respectively). Constructs CCPs 7-8 and 6-8 exhibit measurable affinities for C3b according to surface plasmon resonance, although they are weak compared with CCPs 19-20. Contrary to expectations, none of several constructs encompassing modules from CCP 9 to 15 exhibited significant C3b binding in this assay. Thus, we propose a new functional map of factor H.     

Solution structure and dynamics of the central CCP module pair of a poxvirus complement control protein

The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.     

Immunophysical exploration of C3d-CR2(CCP1-2) interaction using molecular dynamics and electrostatics

The formation of the complex between the d-fragment of the complement component C3 (C3d) and the modular complement receptor-2 (CR2) is important for cross-linking foreign antigens with surface-bound antibodies and C3d on the surface of B cells. The first two modules of CR2, complement control protein modules (CCPs), participate in non-bonded interactions with C3d. We have used computational methods to analyze the dynamic and electrostatic properties of the C3d-CR2(CCP1-2) complex. The interaction between C3d and CR2 is known to depend on pH and ionic strength. Also, the intermodular mobility of the CR2 modules has been questioned before. We performed a 10 ns molecular dynamics simulation to generate a relaxed structure from crystal packing effects for the C3d-CR2(CCP1-2) complex and to study the energetics of the C3d-CR2(CCP1-2) association. The MD simulation suggests a tendency for intermodular twisting in CR2(CCP1-2). We propose a two-step model for recognition and binding of C3d with CR2(CCP1-2), driven by long and short/medium-range electrostatic interactions. We have calculated the matrix of specific short/medium-range pairwise electrostatic free energies of interaction involved in binding and in intermodular communications. Electrostatic interactions may mediate allosteric effects important for C3d-CR2(CCP1-2) association. We present calculations for the pH and ionic strength-dependence of C3d-CR2(CCP1-2) ionization free energies, which are in overall agreement with experimental binding data. We show how comparison of the calculated and experimental data allows for the decomposition of the contributions of electrostatic from other effects in association. We critically compare predicted stabilities for several mutants of the C3d-CR2(CCP1-2) complex with the available experimental data for binding ability. Finally, we propose that CR2(CCP1-2) is capable of assuming a large array of intermodular topologies, ranging from closed V-shaped to open linear states, with similar recognition properties for C3d, but we cannot exclude an additional contact site with C3d.     

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.     

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement-mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein-protein and protein-carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules-the 16th of the 30 modules of complement receptor type 1 (CD35), and the N-terminal module of membrane cofactor protein (CD46)-as well as their solution structures, are compared. Although both examples share broadly similar three-dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module-surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.     

Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains

We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1-4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein. With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6x His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol. A mouse DAF CCP1-4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn(187) and Asn(262) to Gln was also produced. Using Ni(2+)-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6x His tags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody against native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the purified product. Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways. With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained. The production of large quantities of functional recombinant mouse DAF CCP1-4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo.     

Structure-based mapping of DAF active site residues that accelerate the decay of C3 convertases

Focused complement activation on foreign targets depends on regulatory proteins that decay the bimolecular C3 convertases. Although this process is central to complement control, how the convertases engage and disassemble is not established. The second and third complement control protein (CCP) modules of the cell surface regulator, decay-accelerating factor (DAF, CD55), comprise the simplest structure mediating this activity. Positioning the functional effects of 31 substitution mutants of DAF CCP2 to -4 on partial structures was previously reported. In light of the high resolution crystal structure of the DAF four-CCP functional region, we now reexamine the effects of these and 40 additional mutations. Moreover, we map six monoclonal antibody epitopes and overlap their effects with those of the amino acid substitutions. The data indicate that the interaction of DAF with the convertases is mediated predominantly by two patches approximately 13 A apart, one centered around Arg69 and Arg96 on CCP2 and the other around Phe148 and Leu171 on CCP3. These patches on the same face of the adjacent modules bracket an intermodular linker of critical length (16 A.) Although the key DAF residues in these patches are present or there are conservative substitutions in all other C3 convertase regulators that mediate decay acceleration and/or provide factor I-cofactor activity, the linker region is highly conserved only in the former. Intra-CCP regions also differ. Linker region comparisons suggest that the active CCPs of the decay accelerators are extended, whereas those of the cofactors are tilted. Intra-CCP comparisons suggest that the two classes of regulators bind different regions on their respective ligands.     

Synergy between two active sites of human complement receptor type 1 (CD35) in complement regulation: implications for the structure of the classical pathway C3 convertase and generation of more potent inhibitors

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.     

Structural analysis of the complement control protein (CCP) modules of GABA(B) receptor 1a: only one of the two CCP modules is compactly folded

The gamma-aminobutyric acid type B (GABA(B)) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABA(B) receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABA(B) R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMR-derived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABA(B) R1a have strikingly different structural properties, reflecting their different functions.     

Mapping of Eps15 domains involved in its targeting to clathrin-coated pits

Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15 homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly.     

Structural analysis of the C-terminal region (modules 18-20) of complement regulator factor H (FH)

Factor H (FH) is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP) modules of FH (FH18-20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18-20 flexibility in solution using FH18-20 and FH19-20 constructs. In the crystal lattice FH18-20 adopts a "J"-shape: A ~122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18-20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.     

Prediction of the Tertiary Structure of the Complement Control Protein Module

Complement control protein (CCP) modules, or short consensus repeats (SCR), exist in a wide variety of complement and adhesion proteins, principally the selectins. We have predicted the three-dimensional structure of a CCP module based upon secondary structural information derived by two-dimensional NMR [Barlow et al. (1991), Biochemistry 30, 997–1004]. Accordingly, the CCP is predicted to contain seven -strands with extensive hydrogen-bonding interactions, and shows a compact, globular structure. Comparison of this model to the X-ray structure of a kringle domain suggests that the CCP unit is more compact than a kringle structure, and that despite their similarities in size and disulfide bond format, the two are not homologous. Although the function of CCP domains is unknown, it is hoped that the structural model presented herein will facilitate further inquiry into how they contribute to so many systems of biological importance.     

Hotspots organize clathrin-mediated endocytosis by efficient recruitment and retention of nucleating resources

The formation of clathrin-coated pits (CCPs) at the plasma membrane has been reported to sometimes occur repeatedly at predefined sites. However, defining such CCP 'hotspots' structurally and mechanistically has been difficult due to the dynamic and heterogeneous nature of CCPs. Here, we explore the molecular requirements for hotspots using a global assay of CCP dynamics. Our data confirmed that a subset of CCPs is nucleated at spatially distinct sites. The degree of clustering of nucleation events at these sites is dependent on the integrity of cortical actin, and the availability of certain resources, including the adaptor protein AP-2 and the phospholipid PI(4,5)P(2) . We observe that modulation in the expression level of FCHo1 and 2, which have been reported to initiate CCPs, affects only the number of nucleations. Modulation in the expression levels of other accessory proteins, such as SNX9, affects the spatial clustering of CCPs but not the number of nucleations. On the basis of these findings, we distinguish two classes of accessory proteins in clathrin-mediated endocytosis (CME): nucleation factors and nucleation organizers. Finally, we observe that clustering of transferrin receptors spatially randomizes pit nucleation and thus reduces the role of hotspots. On the basis of these data, we propose that hotspots are specialized cortical actin patches that organize CCP nucleations from within the cell by more efficient recruitment and/or retention of the resources required for CCP nucleation partially due to the action of nucleation organizers.     

Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.     

Domain structure and domain-domain interactions in the carboxy-terminal heparin binding region of fibronectin.

The domain structures and stabilities of fragments isolated from the so-called 'hep 2' region of plasma fibronectin have been investigated by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The 30 kDa hep-2A fragment contains three type III modules (III12 to III14), whereas the 40 kDa hep-2B fragment contains four such modules (III12 to III15). Melting of these fragments at neutral pH was irreversible and accompanied by rapid aggregation. In contrast, melting was completely reversible in 50 mM-glycine at pH 2.7, where DSC measurements revealed the presence of three independently folded domains in 30kDa hep-2A and four in 40 kDa hep-2B. That each domain represented a single module was confirmed by measurements with four single-module subfragments, all of which melted reversibly, even at neutral pH. At neutral pH in the presence of 6 M-urea, 30 kDa hep-2A melted reversibly in a sharp peak from which only two transitions could be resolved by deconvolution. Only the larger of these was stabilized by heparin and was assigned to modules III13 and III14. Upon isolation, module III13 melted at lower temperature than in the parent fragment where it is stabilized through an interaction with module III14. We conclude that all type III modules in the hep-2 region of fibronectin constitute independently folded domains. Modules III13 and III14 form a highly co-operative structure through functionally significant interactions that can be disrupted with acid or sufficient concentrations of urea or guanidinium chloride.     

Characterization of the active sites in decay-accelerating factor

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.     

A 3D model for the measles virus receptor CD46 based on homology modeling, Monte Carlo simulations, and hemagglutinin binding studies.

The two terminal complement control protein (CCP) modules of the CD46 glycoprotein mediate measles virus binding. Three-dimensional models for these two domains were derived based on the NMR structures of two CCP modules of factor H. Both CD46 modules are about 35 A long, and form a five-stranded antiparallel beta-barrel structure. Monte Carlo simulations, sampling the backbone torsion angles of the linker peptide and selecting possible orientations on the basis of minimal solvent-exposed hydrophobic area, were used to predict the orientation of CCP-I relative to CCP-II. We tested this procedure successfully for factor H. For CD46, three clusters of structures differing in the tilt angle of the two domains were obtained. To test these models, we mutagenized the CCP modules. Four proteins, two without an oligosaccharide chain and two with mutated short amino acid segments, reached the cell surface efficiently. Only the protein without the CCP-I oligosaccharide chain maintained binding to the viral attachment protein hemagglutinin. These results are consistent with one of our models and suggest that the viral hemagglutinin does not bind at the membrane-distal tip of CD46, but near the concave CCP-II interface region.     

Secondary structure of a complement control protein module by two-dimensional 1H NMR

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Myosin binding protein C positioned to play a key role in regulation of muscle contraction: structure and interactions of domain C1

Myosin binding protein C (MyBP-C) is a thick filament protein involved in the regulation of muscle contraction. Mutations in the gene for MyBP-C are the second most frequent cause of hypertrophic cardiomyopathy. MyBP-C binds to myosin with two binding sites, one at its C-terminus and another at its N-terminus. The N-terminal binding site, consisting of immunoglobulin domains C1 and C2 connected by a flexible linker, interacts with the S2 segment of myosin in a phosphorylation-regulated manner. It is assumed that the function of MyBP-C is to act as a tether that fixes the S1 heads in a resting position and that phosphorylation releases the S1 heads into an active state. Here, we report the structure and binding properties of domain C1. Using a combination of site-directed mutagenesis and NMR interaction experiments, we identified the binding site of domain C1 in the immediate vicinity of the S1-S2 hinge, very close to the light chains. In addition, we identified a zinc binding site on domain C1 in close proximity to the S2 binding site. Its zinc binding affinity (K_d of approximately 10-20 μM) might not be sufficient for a physiological effect. However, the familial hypertrophic cardiomyopathy-related mutation of one of the zinc ligands, glutamine 210 to histidine, will significantly increase the binding affinity, suggesting that this mutation may affect S2 binding. The close proximity of the C1 binding site to the hinge, the light chains and the S1 heads also provides an explanation for recent observations that (a) shorter fragments of MyBP-C unable to act as a tether still have an effect on the actomyosin ATPase and (b) as to why the myosin head positions in phosphorylated wild-type mice and MyBP-C knockout mice are so different: Domain C1 bound to the S1-S2 hinge is able to manipulate S1 head positions, thus influencing force generation without tether. The potentially extensive extra interactions of C1 are expected to keep it in place, while phosphorylation dislodges the C1-C2 linker and domain C2. As a result, the myosin heads would always be attached to a tether that has phosphorylation-dependent length regulation.