FUNCTIONAL-ANALYSIS AND MODELING OF A CONFORMATIONALLY CONSTRAINED ARG-GLY-ASP SEQUENCE INSERTED INTO HUMAN LYSOZYME

To examine the effect of a conformational constraint introduced into t he Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have const ructed mutant proteins by inserting RGD-containing sequences flanked b y two Cys residues between Va174 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast , purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respec tively. In baby hamster kidney cells, these mutants were shown to poss ess high cell adhesion activity by interaction with vitronectin recept or (integrin alpha(v) beta(3)), and this activity is 2-3-fold higher t han that of the RGDS-inserted mutant lysozyme, RGD4. The mutant protei ns also inhibited the binding of human fibrinogen to its receptor (int egrin alpha(IIb)beta(3)) at a lower concentration than the RGD4 protei n. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other witho ut any effects on the mode of the four disulfide bonds present in nati ve human lysozyme. These results suggest that the introduction of a co nformational constraint into the RGD region significantly increases th e cell adhesion activity. The conformation of the RGD region in Cys-RG D4 was modeled by a Monte Carlo simulation. Most of the sampled confor mations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn , and the third has a salt bridge between Arg and Asp. The probability of occurrence in the simulation, as well as the crystal structure of RGD4, suggests that the most probable conformation of RGD belongs to t he first class.