NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48.
The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2).
McAlister
M S
MS
Department of Biochemistry, University of Oxford, UK.
Mott
H R
HR
van der Merwe
P A
PA
Campbell
I D
ID
Davis
S J
SJ
Driscoll
P C
PC
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D018801:CD2 Antigens / Q000737:chemistry*; D000071178:CD48 Antigen; D016466:CHO Cells; D002450:Cell Communication; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011487:Protein Conformation; D051381:Rats; D012995:Solubility
D015703:Antigens, CD; D018801:CD2 Antigens; D000071178:CD48 Antigen; C000604065:CD48 protein, human; C000604066:Cd48 protein, mouse; C000604067:Cd48 protein, rat