Abstract
To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.
Highlights
To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme
The results suggest that the cell adhesion signals in these mutant proteins are transduced to baby hamster kidney (BHK) cells through the interaction with the vitronectin receptor, the integrin Ctvf33'
The x-ray structural analysis, as well as the peptide mapping analysis, indicated that the Cys-RGD4 mutant possesses a new disulfide bond between the two inserted Cys residues, in addition to the four native disulfide bonds of human lysozyme. These results demonstrate that the introduction of a conformational constraint into the RGD sequence of the mutant lysozyme significantly increases the affinity to the integrins, as is the case for an RGD-containing peptide
Summary
To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val and Asn of human lysozyme. The Arg-Gly-Asp (RGD) sequence is a well known site in cell adhesive proteins, such as fibronectin (Pierschbacher and Ruoslahti, 1984), vitronectin (Suzuki et al, 1985), and fibrinogen (Watt et al, 1979), for binding to their receptors, the integrins (Hynes, 1987; Hemler, 1991). In addition to these cell adhesive proteins, a number of other proteins have been found to contain the RGD sequence, but only a limited number of them possess cell adhesion activity. We have already examined the three-dimensional structure of the RGD4 lysozyme by x-ray crystallographic and two-dimensional NMR techniques and shown that the RGD-containing region is conformationally flexible (Yamada et al, 1993)
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