NMR solution structure of a potent cyclic nonapeptide inhibitor of ICAM-1-mediated leukocyte adhesion produced by homologous amino acid substitution.
We have previously described a disulfide-linked cyclic nonapeptide (inhibitory peptide-01, IP01), with the sequence CLLRMRSIC, which binds to intercellular adhesion molecule-1 (ICAM-1), and blocks binding to its counter-structure, the integrin alphaLbeta2 (leukocyte functional antigen-1, LFA-1) (Sillerud et al., J. Peptide Res. 62, 2003: 97). We now report the optimization of this peptide by means of single homologous amino acid substitutions to yield a new peptide (IP02-K6; CLLRMKSAC) which shows an approximately sixfold improvement in inhibitory activity of multivalent leukocyte binding (inhibition constant for 50% inhibition, IC50 = 90 microm) compared with IP01 (IC50 = 580 microm). This improvement in activity gives IP02-K6 potent in vivo activity in a murine model of ischemia reperfusion injury (Merchant et al., Am. J. Physiol. Heart Circ. 284, 2003: H1260). In order to determine the structural features relevant to ICAM-1-binding, we have determined the structure of IP02-K6 using proton nuclear magnetic resonance (NMR) spectroscopy and restrained molecular modeling. In our previously reported study of solution models of IP01, we observed three interconverting conformations during low-temperature molecular dynamics simulation. In the present study, we find a single conformation of IP02-K6 similar to one of the previously found conformations of IP01 (family C). In particular, an R4-S7 beta-turn is present in similar proportions in both conformation C of IP01 and in IP02-K6; this motif is important in binding to ICAM-1 because this turn enables the IP02-K6 backbone to drape over proline-36 on ICAM-1. The NMR-derived solution model of IP02-K6 was found to dock at the alphaLbeta2-binding site on ICAM-1 with no changes in peptide backbone conformation. This docking model displaced five of the 15 alphaLbeta2 residues at the ICAM-1-binding site and provided a rationale for understanding the quantitative relationship between IP02-K6 structure and biologic activity.
Sillerud
L O
LO
Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Burks
E J
EJ
Brown
W M
WM
Brown
D C
DC
Larson
R S
RS
D020816:Amino Acid Motifs; D001665:Binding Sites; D002460:Cell Line; D004220:Disulfides; D006801:Humans; D020128:Inhibitory Concentration 50; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry* / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D013329:Structure-Activity Relationship; D013696:Temperature
D004220:Disulfides; D016169:Lymphocyte Function-Associated Antigen-1; D010455:Peptides; D010456:Peptides, Cyclic; D011522:Protons; C473032:cyclic nonapeptide IP25; D018799:Intercellular Adhesion Molecule-1