Staggered Molecular Packing in Crystals of a Collagen-like Peptide with a Single Charged Pair Rachel Z. Kramer 1 , Manju G. Venugopal 2 , Jordi Bella 1 Patricia Mayville 1 , Barbara Brodsky 2 and Helen M. Berman 1,3 * 1 Department of Chemistry Rutgers University, 610 Taylor Rd, Piscataway, NJ 08854- 8087, USA 2 Department of Biochemistry Robert Wood Johnson Medical School, Piscataway NJ 08855, USA 3 Waksman Institute, Piscataway, NJ 08855, USA The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly) 4 -Glu- Lys-Gly-(Pro-Hyp-Gly) 5 has been determined to 1.75 A Ê resolution. This peptide was designed to examine the effect of a pair of adjacent, oppo- sitely charged residues on collagen triple-helical conformation and inter- molecular interactions. The molecular conformation (a 7 5 triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated NÐH  O1C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water mol- ecules. Solution studies on the EKG peptide indicate stabilization at neu- tral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen ®brils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water- mediated interactions with backbone C  O groups and side-chains. # 2000 Academic Press Keywords: collagen; triple helix; charged residues; staggered packing; hydration *Corresponding author Introduction The triple helix is the principal structural element of collagen and is also an integral feature of a variety of host defense proteins such as the serum complement protein (C1q), mannose-bind- ing protein, and the macrophage scavenger recep- tor (Hoppe & Reid, 1994). The essential structural features of the triple helix were initially elucidated through ®ber diffraction studies of native collagen and synthetic collagen-like polypeptides (Fraser et al., 1979; Rich & Crick, 1961; Yonath & Traub, 1969). The triple helix of collagen is a super-coil formed by the interwinding of three left-handed polyproline II helices in a right-handed manner around a common helical axis. A set of interchain hydrogen bonds in the Rich and Crick collagen II (Rich & Crick, 1961) pattern connects the three chains with a one-residue stagger between neigh- boring chains. Triple helices are characterized by R.Z.K. and M.G.V. contributed equally to this work. Present addresses: M. G. Venugopal, Roche Diagnostics, 235 Hembree Park Drive, Roswell GA 30076.1447, USA; J. Bella, Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK. Abbreviations used: rms, root-mean-square; PPG 2, polymer-like structure of the triple-helical peptide (Pro- Pro-Gly) 10 determined to 1.75 A Ê resolution (Kramer et al., 1998); Gly!Ala, the triple-helical peptide (Pro-Hyp- Gly) 4 -Pro-Hyp-Ala-(Pro-Hyp-Gly) 5 determined by Bella et al. (1994); Hyp, hydroxyproline; EKG, the triple- helical peptide (Pro-Hyp-Gly) 4 -Glu-Lys-Gly-(Pro-Hyp- Gly) 5 ; T3-785, the triple-helical peptide, (Pro-Hyp-Gly) 3 - Ile-Thr-Gly-Ala-Arg-Gly-Pro-Hyp-Gly-(Pro-Hyp-Gly) 3 . E-mail address of the corresponding author: berman@rcsb.rutgers.edu doi:10.1006/jmbi.2000.4017 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 301, 1191±1205 0022-2836/00/051191±15 $35.00/0 # 2000 Academic Press