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

Abstract

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.Keywords: complement system; C3d; factor H; Poisson-Boltzmann electrostatics; molecular dynamics; steered molecular dynamics; MM/GBSA binding free energy Statement: Crystallographic structures propose three binding sites for the complex of immune system protein C3d with the regulator factor H 19-20. Since crystallization of proteins can perturb physiological protein structure, we computationally characterize the interactions at the three putative binding sites. Our studies contribute to understanding the physicochemical basis of FH 19-20 interactions with C3d and provide a molecular basis for understanding autoimmune diseases such as atypical hemolytic syndrome.Abbreviations: AP, alternative pathway; C3, complement component 3; C3b, complement component 3b; FB, factor B; C3a, complement component C3a; C3d, complement component 3d; FH, factor H; iC3b, proteolytically-inactive complement component 3b; CCP, complement control protein; aHUS, atypical hemolytic uremic syndrome; PDB, protein data bank; GAG, glycosaminoglycan; CR2, complement receptor 2; AESOP, analysis of electrostatic similarities of proteins; WT, wild type; MD, molecular dynamics; RMSD, root-mean-square deviation; SASA, solvent-accessible surface area; SMD, steered molecular dynamics; MM/GBSA, molecular mechanics/generalized Born surface area.Additional Supporting Information may be fo...

Structural insights on complement activation

“…MCP, DAF and CR1 are thought to follow similar mechanisms [4,5]. In addition, SCR19 and SCR20 in FH contain a C3b/polyanion binding site implicated in preventing AP activation on host cells [10][11][12]. In conclusion, at the same time that activation of C3 to generate C3b allows formation of the C3-convertase and triggers the amplification loop, it also generates the way to be regulated.…”

Immunophysical Evaluation of the Initiating Step in the Formation of the Membrane Attack Complex

“…Thus, to elucidate on how C3d and C5d achieve their versatile functions, we performed a comparative analysis of the physicochemical properties of C3d and C5d, also including a comparative analysis of C3d and C4d for completion. Earlier studies have identified the residues of C3d that interact with FH [29] and CR2 [28]. FH is a potent regulator of C3b, and C3b's convertase complexes, that inhibits opsonophagocytosis on host cells.…”

“…The viral vaccinia control protein (VCP), that is structurally and functionally homologous to SPICE, is also expected to have a similar binding mode to C3b. In addition, the stand-alone C3d domain, is known to interact with modules CCP1-2 of complement receptor 2 (CR2) [28], modules CCP19-20 of FH [29], in addition to modules CCP1-4 (mentioned above as interacting along C3b), and S. aureus proteins Efb-C, Ecb, and Sbi [30,31]. These structural observations make the C3d domain multifunctional in interacting with complement natural and viral regulators, when C3d is part of C3b, and in attracting CR2, FH (CCP19-20), and bacterial regulators when C3d is stand-alone.…”

AESOP: A Python Library for Investigating Electrostatics in Protein Interactions

“…The alanine scan is a perturbation method of identifying individual amino acids that have a significant effect in the formation of the parent protein complex (loss of binding mutations). It can also identify mutations or sites of mutations that will enhance association (gain of binding mutations) in studies of design of protein-protein interfaces (29)(30)(31).…”