Multi‐scale characterization of the energy landscape of proteins with application to the C3D/Efb‐C complex

Abstract

We present a novel multi-level methodology to explore and characterize the low energy landscape and the thermodynamics of proteins. Traditional conformational search methods typically explore only a small portion of the conformational space of proteins and are hard to apply to large proteins due to the large amount of calculations required. In our multi-scale approach, we first provide an initial characterization of the equilibrium state ensemble of a protein using an efficient computational conformational sampling method. We then enrich the obtained ensemble by performing short Molecular Dynamics (MD) simulations on selected conformations from the ensembles as starting points. To facilitate the analysis of the results we project the resulting conformations on a low-dimensional landscape to efficiently focus on important interactions and examine low energy regions. This methodology provides a more extensive sampling of the low energy landscape than an MD simulation starting from a single crystal structure as it explores multiple trajectories of the protein. This enables us to obtain a broader view of the dynamics of proteins and it can help in understanding complex binding, improving docking results and more. In this work we apply the methodology to provide an extensive characterization of the bound complexes of the C3d fragment of human Complement component C3 and one of its powerful bacterial inhibitors, the inhibitory domain of Staphylococcus aureus extra-cellular fibrinogenbinding domain (Efb-C) and two of its mutants. We characterize several important interactions along the binding interface and define low free energy regions in the three complexes.

Advances in Understanding the Structure, Function, and Mechanism of the SCIN and Efb Families of Staphylococcal Immune Evasion Proteins

“…While the contributions of specific positions in C3d have not yet been examined by mutagenesis, this type of detailed structure/function analysis has been carried out for Efb-C (Hammel et al 2007b; Haspel et al 2010; Haspel et al 2008). This work has demonstrated that positions R131 and N138 of Efb-C are particularly critical determinants for both the affinity and stability of the C3d complex.…”

“…Studies on both single and pairwise substitutions at positions 131 and 138 revealed that interactions between Efb-C and C3d are dependent upon both the charge and polar nature of these side chains. Whereas the observed K D rose to 26 and 19 nM, respectively, when either residue alone was mutated to alanine (Haspel et al 2010; Haspel et al 2008), the corresponding double mutant had no residual affinity (Hammel et al 2007b; Haspel et al 2008). Furthermore, while loss of the side chain functionality had only minor effects on the ionic strength-dependent association phase, the rate of complex dissociation was greatly accelerated in each mutant (Haspel et al 2008).…”

Complement Inhibition by Staphylococcus aureus: Electrostatics of C3d–EfbC and C3d–Ehp Association

“…14 Furthermore, later studies thoroughly investigated the characteristics of these mutants in comparison to wild-type EfbC. 15,16 In accordance with the two-step binding model of protein association, the results of surface plasmon resonance experiments indicated that these mutants have little effect on association rates, but drastically increase dissociation rates for the complex with C3d. This indicates that highly-specific interfacial interactions are responsible for maintaining complex stability, while complex association is governed by the overall electrostatic potential distributions of C3d and EfbC.…”