SOLUTION STRUCTURE OF A PEPTIDE FROM A PHAGE DISPLAY LIBRARY USING ANTI-CARDIOLIPIN ANTIBODY: A b2GPI EPITOPE MIMIC.

Sem, D.S., Victoria, E.J., Marquis, D.M., Pabarcus, M.K., Coutts, S.M.

La Jolla Pharmaceutical Company, San Diego, CA 92121

Random peptide phage display is a powerful tool for the selection of ligands with high-affinity for receptors like enzymes and antibodies. Two peptides were isolated from phage library screens using anti-cardiolipin antibody (ACA) from a patient with SLE and recurrent thrombosis. Although the original peptides had a Pro in the second to last position, this amino acid was removed since 2D DQF-COSY NMR data suggested the presence of two different structures from the cis and trans isomers at this position. Removal of the Pro gave peptides with Kds in the 50-100 nM range for binding to ACA, and the expected number of peaks in the fingerprint region of the DQF-COSY spectrum. The resulting cyclic peptides are 5A12: GPCLILAPDRCG, and CB2: GPCILLARDRCG. The major difference between these peptides is the substitution of Arg for Pro8, which results in much less dispersion in the 1D 1H NMR spectrum of CB2, consistent with 5A12 being a more rigid peptide. This Arg substitution also produces a 0.55 kcal/mol stabilization of the ionized Asp9, and only 0.23 kcal/mol stabilization of the ionized carboxy terminus, as reflected in pKa values. A structural analysis was carried out on the more ordered 5A12 peptide. Although deuterium exchange and temperature coefficient data show no evidence of hydrogen binding, the NOE, ROE and coupling data are consistent with one structure. Distance geometry calculations yielded a family of 50 structures. The 15 best had a mean RMSD for all atoms of 2.1 ± 0.2 angstroms. The peptide has an oval shape with turns at opposite ends of the molecule: at the disulfide, and at Pro8, which is cis. There is also a kink in the backbone in the LIL region. Finally, the carboxy terminal Gly is very mobile, as it is the only residue with a positive intraresidue NOE. The 5A12 peptide is a mimic of the as yet unidentified ACA epitope on b2GPI, and this represents the first structural information on such a mimotope.

The structure was determined for 5A12, which lacks the carboxy-terminal proline. Numbering is: G1P2C3L4I5L6A7P8D9R10C11G12.

Presented at the American Society for Biochemistry and Molecular Biology meeting, June 2-6, 1996, New Orleans, LA.