Thermodynamic Studies on the Interaction of the Third Complement Component and Its Inhibitor, Compstatin

Abstract

Compstatin is a 13-residue cyclic peptide that inhibits complement activation by binding to complement component, C3. Although the activity of compstatin has been improved severalfold using combinatorial and rational design approaches, the molecular basis for its interaction with C3 is not yet fully understood. In the present study, isothermal titration calorimetry was employed to dissect the molecular forces that govern the interaction of compstatin with C3 using four different compstatin analogs. Our studies indicate that the C3-compstatin interaction is an enthalpy-driven process. Substitution of the valine and histidine residues at positions 4 and 9 with tryptophan and alanine, respectively, resulted in the increase of enthalpy of the interaction, thereby increasing the binding affinity for C3. The data also suggest that the interaction is mediated by water molecules. These interfacial water molecules could be the source for unfavorable entropy and large negative heat capacity changes observed in the interaction. Although part of the negative heat capacity changes could be accounted for by the water molecules, the rest might be resulting from the conformational changes in C3 and/or compstatin up on binding. Finally, we propose based on the pK a values determined from the protonation studies that histidine on compstatin participates in protonation changes and contributes to the specificity of the interaction between compstatin and C3. These protonation changes vary significantly between the binding of different compstatin analogs to C3.

Herpes Simplex Virus Type 1 and 2 Glycoprotein C Prevents Complement-Mediated Neutralization Induced by Natural Immunoglobulin M Antibody

“…High concentrations of C3 present in NHS make it difficult to deplete; therefore, we used compstatin to inhibit C3 activation and determine whether C3 is necessary to neutralize gC-null viruses. Compstatin (4W9A) is a small synthetic peptide that interferes with complement at low concentrations by binding C3, preventing its activation (29,30,45). Experiments were performed to examine neutralization following treatment with 20% NHS or 20% NHS treated with either active (4W9A) or (Fig.…”

“…Serum to inactivate the classical and mannan-binding lectin pathways (20); and 100 mM D-mannose to interfere with activation of the mannan-binding lectin pathway (9,28). To interfere with C3 activation, NHS was treated with the small synthetic peptide compstatin (4W9A) at a concentration of 40 M, while 40 M linear compstatin was used as an inactive control (29,30,45).…”

Compstatin: A Complement Inhibitor on its Way to Clinical Application

“…Biophysical data confirmed a single binding site for compstatin on C3, while deviation from a simple kinetic 1:1 model in SPR indicated a conformational rearrangement of the peptide and/or protein upon binding (Katragadda et al 2004;Sahu et al 2000). Later, the binding region could be narrowed down to the C-terminal 40-kDa part of C3, which includes several macroglobulin domains (MG 3-6 β ) as well as the linker (LNK) C3b, and C3c (Janssen et al 2006;Wiesmann et al 2006) offered an unprecedented insight into the complement activation process.…”

“…Further thermodynamic analyses indicated that the C3-compstatin interaction is largely driven by enthalpic contributions. In this context, the beneficial effects of a valine-totryptophan substitution at position 4 (and also histidine-to-alanine at position 9) could be linked to an increased enthalpy and binding affinity to C3 (Katragadda et al 2004). …”

Structure-Based Integrative Computational and Experimental Approach for the Optimization of Drug Design

“…Besides IC 50 activity measurements that were performed for each synthesized analog, kinetic and thermodynamic binding studies were performed using surface plasmon resonance [12,14], and isothermal titration calorimetry [15]. The structural, binding, and activity studies were useful to form testable structure-binding and structure-activity hypotheses.…”