STRUCTURAL CHARACTERIZATION AND OPTIMIZATION OF ANTIBODY-SELECTED PHAGE LIBRARY MIMOTOPES OF AN ANTIGEN ASSOCIATED WITH AUTOIMMUNE RECURRENT THROMBOSIS

The presence of high titers of anti-cardiolipin antibodies (ACA's) of autoimmune origin, which are known to bind to plasma beta(2)-glycoprot ein I (aka apolipoprotein H) correlates clinically with autoimmune rec urrent thrombosis. Soluble beta(2)-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a K-d Of 1 .4 mu M, but if the reactants are reversed and beta(2)-glycoprotein I is on the solid-phase support, then the K-d is 52 nM. This 27-fold dif ference in affinity reflects the avidity/entropic advantage obtained f or an antibody binding to an antigen that is made multivalent because it is attached to a solid phase. A mimotope of this antigen, selected from a phage display peptide library screen with an ACA, has been show n to bind to solid-phase ACA as a phage, using surface plasmon resonan ce. This peptide is representative of the motif from 37 peptides obtai ned in a previously reported phage library screen with this ACA (I). A synthetic version of this peptide, referred to as P4, has the sequenc e: C(4)I(5)L(6)L(7)A(8)R(9)D(10)R(11)C(12)P(13)G(14), and binds to its selecting antibody with a K-d Of 42 nM. NMR data indicate that prolin e-13 is present in both cis and trans configurations, and that these t wo geometries dramatically affect the overall tertiary structure of th e molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having lo w affinity for binding ACA. Replacement of the arginine-9 position wit h a proline decreases binding affinity to ACA 10-fold. Another phage l ibrary-selected peptide has a proline in position 9, but also has a le ucine in position 5, instead of isoleucine. Since its affinity for ACA is nearly as good as that for peptide P4, the phage library screening must have selected for a non-beta-branched amino acid in this positio n to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version of the ant ibody-selected phage peptide P4 with the central proline was determine d. This peptide has one turn comprised of Ala-Pro-Asp-Arg, with the pr oline peptide bond in the cis configuration, and another turn that con tains the disulfide and adjacent residues. If the disulfide is replace d by a thioether, and the central proline by an alpha-methyl proline, in an attempt to make the peptide more biologically stable, there is l ittle adverse effect on affinity for ACA. The thioether bond/turn is f airly well defined with a Ca to Cor separation of 4.9 +/- 0.8 Angstrom . The alpha-methyl proline adopts the trans configuration, and this ce ntral Ala-(alpha-methyl-Pro)-Asp-Arg turn adopts a distorted type I tu rn conformation with a probable i to i+3 hydrogen bond. Modeling studi es suggest that the proline peptide bond configuration switched from c is to trans in the presence of the alpha-methyl,group on proline becau se of steric hindrance with the beta-carbon of the preceding residue. Overall, this peptidomimetic molecule is structurally very similar to the peptide with natural amino acids, with an rmsd difference of only 1.37 Angstrom, when comparing backbone atoms.