Use of phage display to probe the evolution of binding specificity and affinity in integrins.
The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.
Li
Renhao
R
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Hoess
Ronald H
RH
Bennett
Joel S
JS
DeGrado
William F
WF
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016023:Integrins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D016023:Integrins; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D000516:alpha-Amylases; C030526:tendamistate