Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.
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.
Yamada
T
T
Protein Engineering Research Institute, Osaka, Japan.
Song
H
H
Inaka
K
K
Shimada
Y
Y
Kikuchi
M
M
Matsushima
M
M
D001216:Asparagine; D001483:Base Sequence; D003196:Computer Graphics; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry*; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014633:Valine
D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011994:Recombinant Proteins; D001216:Asparagine; C047981:arginyl-glycyl-aspartic acid; D009113:Muramidase; D014633:Valine