Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies.
A general strategy is presented here for producing glycan-free forms of glycoproteins without loss of function by employing apolar-to-polar mutations of surface residues in functionally irrelevant epitopes. The success of this structure-based approach was demonstrated through the expression in Escherichia coli of a soluble 11 kDa adhesion domain extracted from the heavily glycosylated 55 kDa human CD58 ectodomain. The solution structure was subsequently determined and binding to its counter-receptor CD2 studied by NMR. This mutant adhesion domain is functional as determined by several experimental methods, and the size of its binding site has been probed by chemical shift perturbations in NMR titration experiments. The new structural information supports a 'hand-shake' model of CD2-CD58 interaction involving the GFCC'C" faces of both CD2 and CD58 adhesion domains. The region responsible for binding specificity is most likely localized on the C, C' and C" strands and the C-C' and C'-C" loops on CD58.
Sun
Z Y
ZY
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Dötsch
V
V
Kim
M
M
Li
J
J
Reinherz
E L
EL
Wagner
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D018801:CD2 Antigens / Q000737:chemistry / Q000378:metabolism; D018968:CD58 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D018801:CD2 Antigens; D018968:CD58 Antigens; D010446:Peptide Fragments; D011134:Polysaccharides; D011994:Recombinant Proteins