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

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 ␤-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the ␤-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 ␤-turn. 2) The type I ␤-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I ␤-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the ␤-turn is essential for activity. 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, 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) ␤-Turn residues

Studies of Structure-Activity Relations of Complement Inhibitor Compstatin

“…Additional NMR and inhibitory activity studies of seven compstatin analogs designed to enhance or disrupt structure and/or function were used as an aid to determine which structural elements are important for the structural stability and inhibitory activity of compstatin (12). Based on these studies, we proposed that the four residues of the ␤-turn and the disulfide bridge between Cys 2 -Cys 12 are essential for both structural stability and inhibitory activity of compstatin. We also proposed that three of six residues of the hydrophobic cluster are essential for inhibitory activity of compstatin, with residues Cys 2 , Cys 12 , and Val 3 being indispensable for inhibition (8,10,12).…”

“…Based on these studies, we proposed that the four residues of the ␤-turn and the disulfide bridge between Cys 2 -Cys 12 are essential for both structural stability and inhibitory activity of compstatin. We also proposed that three of six residues of the hydrophobic cluster are essential for inhibitory activity of compstatin, with residues Cys 2 , Cys 12 , and Val 3 being indispensable for inhibition (8,10,12). The aim of the present study is to understand the structurefunction relations of compstatin and to optimize the amino acids outside the disulfide bridge, Val 3 , and ␤-turn that contribute to the inhibitory activity of compstatin.…”

“…An Ala scan indicated that the amino acids Cys 2 , Val 3 , and Cys 12 of the hydrophobic cluster and the amino acids of the ␤-turn are essential for the activity of compstatin in vitro (8,10). Studies of the inhibitory activity of a linear compstatin analog, where Cys 2 and Cys 12 were reduced and alkylated, or analogs with reduced length between the two cysteines resulted in inactive peptides (8,10). Additional NMR and inhibitory activity studies of seven compstatin analogs designed to enhance or disrupt structure and/or function were used as an aid to determine which structural elements are important for the structural stability and inhibitory activity of compstatin (12).…”

“…Studies of the inhibitory activity of a linear compstatin analog, where Cys 2 and Cys 12 were reduced and alkylated, or analogs with reduced length between the two cysteines resulted in inactive peptides (8,10). Additional NMR and inhibitory activity studies of seven compstatin analogs designed to enhance or disrupt structure and/or function were used as an aid to determine which structural elements are important for the structural stability and inhibitory activity of compstatin (12). Based on these studies, we proposed that the four residues of the ␤-turn and the disulfide bridge between Cys 2 -Cys 12 are essential for both structural stability and inhibitory activity of compstatin.…”

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

“…The computational methods were hybrid distance geometry/simulated annealing [11] and global optimization [3]. Subsequently, optimization of the sequence of compstatin was performed using rational design based on NMR structural studies (but not computational complete structure determination) and structure-activity correlations, which yielded several active analogs with up to 4-fold higher inhibitory activity than the parent peptide [6]. The rational design determined that 7 of the 13 amino acids of compstatin were indispensable for activity, and provided the following sequence template for further optimization: Ac-X[CVXQDWGXXXC]X-NH 2 (called active sequence template), where the 6 amino acids marked with X were optimizable [5].…”