STRUCTURE 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 constructe d a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val(74) and Asn(75) of human lysozyme, The CR GDSC-inserted lysozyme was expressed in yeast, purified, and designate d as Cys-RGD4, Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inser ted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a ly sozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional s tructure was determined at 1.6-Angstrom resolution by x-ray crystallog raphy. In contrast to RGD4, the inserted RGD-containing region of Cys- RGD4 was well defined, The structural analysis revealed that the two i nserted Cys residues form a new disulfide bond in Cys-RGD4, as expecte d, and that the RGD region assumes a type II' beta-turn conformation o f Gly-Asp with a hydrogen bond between the C=O of Arg and the H-N of S er. In addition, it was confirmed that two more hydrogen bonds are pre sent in the RGD region of the Cys-RGD4 lysozyme, These results suggest that the conformation of the RGD-containing region is rigid and stabl e in the Cys-RGD4 molecule and that the type II' beta-turn structure o f RGD is essential for binding to integrins with high affinity.