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 beta2-glycoprotein I (aka apolipoprotein H), correlates clinically with autoimmune recurrent thrombosis. Soluble beta2-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a Kd of 1.4 microM, but if the reactants are reversed and beta2-glycoprotein I is on the solid-phase support, then the Kd is 52 nM. This 27-fold difference in affinity reflects the avidity/entropic advantage obtained for 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 shown to bind to solid-phase ACA as a phage, using surface plasmon resonance. This peptide is representative of the motif from 37 peptides obtained in a previously reported phage library screen with this ACA (1). A synthetic version of this peptide, referred to as P4, has the sequence: A1G2P3C4I5L6L7A8R9D10R11C12P13G14, and binds to its selecting antibody with a Kd of 42 nM. NMR data indicate that proline-13 is present in both cis and trans configurations, and that these two geometries dramatically affect the overall tertiary structure of the molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having low affinity for binding ACA. Replacement of the arginine-9 position with a proline decreases binding affinity to ACA 10-fold. Another phage library-selected peptide has a proline in position 9, but also has a leucine 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 position to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version of the antibody-selected phage peptide P4 with the central proline was determined. This peptide has one turn comprised of Ala-Pro-Asp-Arg, with the proline peptide bond in the cis configuration, and another turn that contains the disulfide and adjacent residues. If the disulfide is replaced by a thioether, and the central proline by an alpha-methyl proline, in an attempt to make the peptide more biologically stable, there is little adverse effect on affinity for ACA. The thioether bond/turn is fairly well defined with a Calpha to Calpha separation of 4.9 +/- 0.8 A. The alpha-methyl proline adopts the trans configuration, and this central Ala-(alpha-methyl-Pro)-Asp-Arg turn adopts a distorted type I turn conformation with a probable i to i+3 hydrogen bond. Modeling studies suggest that the proline peptide bond configuration switched from cis to trans in the presence of the alpha-methyl group on proline because 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 A, when comparing backbone atoms.
Sem
D S
DS
La Jolla Pharmaceutical Company, San Diego, California 92121, USA.
Baker
B L
BL
Victoria
E J
EJ
Jones
D S
DS
Marquis
D
D
Yu
L
L
Parks
J
J
Coutts
S M
SM
D017153:Antibodies, Anticardiolipin / Q000737:chemistry / Q000378:metabolism*; D001324:Autoantigens / Q000737:chemistry* / Q000276:immunology; D001327:Autoimmune Diseases / Q000276:immunology / Q000378:metabolism*; D001435:Bacteriophages / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D003198:Computer Simulation; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D018716:Molecular Mimicry; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D011392:Proline / Q000378:metabolism; D012008:Recurrence; D012996:Solutions; D013927:Thrombosis / Q000276:immunology / Q000378:metabolism*
D017153:Antibodies, Anticardiolipin; D001324:Autoantigens; D000939:Epitopes; D009842:Oligopeptides; D019151:Peptide Library; D012996:Solutions; D011392:Proline