J. Mol. Biol. (1988) 201, 625-636 Solution Structures of the Rabbit Neutrophil Defensin NP-5 Arthur Pardi’t, Dennis R. Hare2, Michael E. Selsted3 Robert D. Morrison2, Donna A. Bassolino’ and Alvin C. Bach, IIlf 1Department of Chemistry Rutgers, The State University of New Jersey New Brunswick, NJ 08903, U.S.A. ’ Hare Research, Inc. 14810 216th Avenue, N.E. Woodinville, WA 98072, U.S.A. 3 Departments of Medicine and Pathology The Center for Health Science University of California, Los Angeles, CA 90024, U.S.A. (Received 14 October 1987, and in revised form 2 January 1988) Solution structures of the rabbit neutrophil defensin NP-5 have been determined by ‘H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter- residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo- specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined. 1. Introduction Progress in two-dimensional n.m.r.5 techniques and computational methods have made it possible to determine the solution structure of biopolymers t Present address and where correspondence should be sent: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 30309, U.S.A. $ Present address: Medical Products Department, E.I. du Pont, Wilmington, DE 19898, U.S.A. Q Abbreviations used: n.m.r., nuclear magnetic resonance; NOE, nuclear Overhauser effect; NP-1, NP-2, NP-4 and NP-5, neutrophil peptides 1, 2, 4 and 5, respectively, from rabbit; COSY, 2-dimensional correlation spectroscopy; FID, free induction decay; NOESY, 2-dimensional nuclear Overhauser effect spectroscopy; p.p.m., parts per million; r.m.s., root- mean-squared. with a precision that approaches X-ray crystallo- graphy (for a review, see Wiithrich, 1986). Recently, the X-ray crystal structure and the n.m.r. solution structure of the small protein cc-amylase inhibitor Tendamistat were indepen- dently determined (Kline et al., 1986; Pflugrath et al., 1986). This study showed that at the level of the backbone folding of the protein, both techniques produced basically the same structure. The com- bination of the proton-proton internuclear distances derived from two-dimensional NOE experiments with computational methods such as a distance geometry algorithm (Crippen, 1977; Kuntz et al., 1979; Have1 et al., 1979; Braun et al., 1981; Braun & Go, 1985; Have1 & Wiithrich, 1985; Clore et al., 1986a), restrained molecular dynamics or restrained molecular mechanics programs (Kaptein et al., 1985; Clore et al., 1985, 1986a,6; Bruenger et al., 1986; Fesik et al., 1986; Jeffs et al., 1986; Holak 0022-2836/88/110625-12 $03,00/O 625 0 1988 Academic Press Limited