Identification of the c3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B¹

Abstract

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH ₃) in the presence of Mg²⁺. A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH₃), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH₃) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH ₃). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central β-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A β-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.

Introduction

The complement system is an important component of immune defence against infection, comprising over 30 proteins which function as enzymes, binding proteins, regulators or membrane-bound receptors. It is part of the innate (non-adaptive) immune system which responds to challenges by micro-organisms before an adaptive response has developed (Law & Reid, 1995). Factor B is a key component of the alternative pathway of complement activation. It is cleaved into the Ba and Bb fragments in the presence of C3b, Mg²⁺ and factor D. This leads to the formation of the alternative pathway C3 convertase, the complex between C3b and Bb, that converts C3 into C3b and C3a. The formation of C3b leads to the exposure of the thiolester group which is buried in native C3, and its deposition on an appropriate surface initiates a rapid positive amplification loop. C3u is another activated form of C3 in which the internal thiolester is hydrolysed by water, and C3u undergoes a conformational change like that found in C3b.

Factor B contains five domains. The N-terminal three domains are short consensus/complement repeat (SCR; also known as complement control protein) domains that correspond to the Ba fragment, and the remaining two are a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain in the Bb fragment. vWF-A domains (also known as “adhesive” or A-domains and “inserted” or I-domains) occur in many cell surface receptor proteins and proteins of the extracellular matrix. The vWF-A protein fold was first identified by prediction analyses that revealed that it was similar to that of a dinucleotide-binding fold with amphiphatic α-helices surrounding a central hydrophobic, largely parallel six-stranded β-sheet Edwards and Perkins 1995, Edwards and Perkins 1996. Since then, 14 crystal structures have been solved for five different vWF-A domains, namely complement receptor type 3 (CR3; CD11b), lymphocyte function-associated antigen-1 (LFA-1; CD11a), very late activation protein receptor-2 (VLA-2; CD49b), and the A1 and A3 domains of von Willebrand factor Lee et al 1995a, Lee et al 1995b, Baldwin et al 1998, Qu and Leahy 1995, Qu and Leahy 1996, Emsley et al 1997, Emsley et al 1998, Celikel et al 1998, Huizinga et al 1997, Bienkowska et al 1997. Factor B, C2, CR3, and complement receptor type 4 are four complement proteins that each contain a vWF-A domain. As all four interact with various forms of C3 or C4 (a homologue of C3), in which C3b and C3u are the activated forms of C3, and iC3b is a cleaved form of C3b, it is likely that the vWF-A domain mediates this ligand binding with C3 or C4. In confirmation of this, the vWF-A domain of factor B binds to C3b (Williams et al., 1999), and mutagenesis of residues in the vWF-A domain of intact factor B affects its binding to C3b Tuckwell et al 1997, Hourcade et al 1999. Competition binding studies have demonstrated that both Ba and Bb fragments can separately inhibit the binding of factor B to immobilised C3u (Williams & Sim, 1994). The SCR domains in the Ba fragment interact with C3b or C3u Hourcade et al 1995, Pryzdial and Isenman 1988, Ueda et al 1987. The SP domain also demonstrates C3b binding activity Lambris and Muller-Eberhard 1984, Sanchez-Corral et al 1990. Despite the evidence for these protein-protein interactions, structural knowledge of the residues responsible for these is presently incomplete.

The SP domain of factor B is the site of catalytic action of the C3 convertase of the alternative pathway. This SP domain differs from the digestive and blood coagulation serine proteases by possessing a sizeable vWF-A domain at its N terminus. The cleavage leading to the activation of factor B occurs at the N terminus of the vWF-A domain, in contrast to the cleavage of small N-terminal activation peptides in these other SP domains. The clarification of the relationship between the vWF-A and SP domains in factor B is therefore of great interest.

Matrix-assisted laser desorption ionisation mass spectrometry is a powerful method for the investigation of protein-protein interactions Beavis and Chait 1996, Krone et al 1997. In application to proteins and peptides, a matrix compound such as sinapinic acid is used to assist the vapourization of these from a chip surface, and the mass spectrometer uses the difference in mass-to-charge ratio (m/z) to separate these and determine their masses to high accuracies (Krone et al., 1997). This accuracy is sufficient to identify the peptide sequences in question. Matrix-assisted laser desorption ionisation mass spectrometry has enabled proteolytic fragmentation patterns to be identified in protein-protein complexes (Beavis & Chait, 1996), and this provides structural information on the residues that participate at the interface between the two proteins in the complex. The use of an activated chip surface to which one of the two proteins can be bound covalently simplifies the preparation and identification of these proteolytic fragments (Spencer et al., 1999). This method is known as surface-enhanced laser desorption ionisation affinity mass spectrometry (SELDIAMS) Hutchens and Yip 1993, Kuwata et al 1998, Leung et al 1998. Here, we describe the application of SELDIAMS to study the interactions between C3b and factor B based on a recombinant vWF-A domain, the native Ba and Bb fragments and native factor B. The interpretation of the peptide data using a homology model for the factor B vWF-A domain based on the knowledge of 14 homologous crystal structures for five different vWF-A domains enabled us to demonstrate that the active site crevice at the carboxyl edge of the β-sheet structure of the vWF-A domain binds directly to C3b, and it was inferred that the amino edge of this β-sheet provides the connections with the SCR and SP domains of factor B. The implications of these observations are discussed.