The Structural Basis of Compstatin Activity Examined by Structure-Function-based Design of Peptide Analogs and NMR

Dimitrios Morikis,Melinda Roy,Arvind Sahu,Anastasios Troganis,Patricia A Jennings,George C Tsokos,John D Lambris

doi:10.1074/jbc.m200021200

Abstract

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Highlights

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b
We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin
The following have been shown. (a) Compstatin inhibits complement activation in vitro in human serum [8]. (b) Compstatin totally inhibits in vivo heparin/protamine-induced complement activation in primates without side effects, in a situation that is typical in cardiac surgery [9]. (c) Compstatin inhibits complement activation without toxicity in whole blood, in models of extracorporeal circuits, which resemble those used in cardiopulmonary bypass, dialysis, and plasmapheresis [10]. (d) Compstatin prolongs the lifetime of porcine-to-human ex vivo perfused kidney xenograft model with human blood [11]. (e) compstatin does not appear to have significant cytotoxicity as it showed little or no inhibition of clotting [12]

Summary

Introduction

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. 5) The type I ␤-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, aiding in the formation of the hydrophobic cluster. Complement is part of the innate immune system while acting as a bridge between the innate and adaptive immune systems, and its activation is an important line of defense against invading foreign pathogens [3,4,5], its inappropriate activation can result in host cell damage [6] This is the case in more than 25 pathological conditions, including a number of autoimmune diseases, burn injuries, ischemia reperfusion injuries, stroke, dialysis, and cardiopulmonary bypass (see Ref. 7 for a complete list and references). The following have been shown. (a) Compstatin inhibits complement activation in vitro in human serum [8]. (b) Compstatin totally inhibits in vivo heparin/protamine-induced complement activation in primates without side effects, in a situation that is typical in cardiac surgery [9]. (c) Compstatin inhibits complement activation without toxicity in whole blood, in models of extracorporeal circuits, which resemble those used in cardiopulmonary bypass, dialysis, and plasmapheresis [10]. (d) Compstatin prolongs the lifetime of porcine-to-human ex vivo perfused kidney xenograft model with human blood [11]. (e) compstatin does not appear to have significant cytotoxicity as it showed little or no inhibition of clotting [12]

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