STABILIZATION OF AN ISOLATED HELICAL CAPPING BOX IN SOLUTION BY HYDROPHOBIC INTERACTIONS - EVIDENCE FROM THE NMR-STUDY OF BIOACTIVE PEPTIDES FROM THE C-TERMINUS OF HUMAN C5A ANAPHYLATOXIN

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) J ournal of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance e xperiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) -Phe67- Lys68-Asp69-Met70-Gln71-Leu72-Gly73-Arg74). Medium-range nuclear Overh auser effects (NOEs) were observed for residues 65-70 of this C5a pept ide, suggesting that this region adopts a folded conformation in a sig nificant population of the solution conformational ensemble. Quantitat ive analyses of (3)J(NH-alpha H) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65 % in the regio n Ser66-Met70. Additional evidence supporting the presence of a helica l turn includes reduced amide,proton temperature coefficients and lowe red (3)J(HN-alpha H) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The si de chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic c luster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH grou ps of these two, residues, indicating that residues Ser66-Phe67-Lys68- Asp69 (or SFKD) form a helix-stablizing capping box [E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605, H. X. Zhou et al. (1 994) Proteins: Structure, Function and Genetics, Vol. 18, p. I] even w ithin the single turn of helical structure found in the analogue Cia p eptide. A comparison of the nmr results obtained for the analogue pept ide and the natural decapeptide C5a 65-74 6-His67-Lys68-Asp69-Met70-Gl n71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues S er66-Asp69 in the C5a peptides through more extensive hydrophobic/arom atic interactions between residues Tyr65, Phe67, and Met70 in the anal ogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the fi rst experimental demonstration of hydrophobic determinants in helical capping-box interactions proposed recently, by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stablized helical turn may also account for th e greater. potency of the analogue peptide C5a 65-74 (Tyr65, Phe67) in receptor-binding assays. (C) 1996 John Wiley & Sons, Inc.