McAlister M S, Mott H R, van der Merwe P A, Campbell I D, Davis S J, Driscoll P C
Department of Biochemistry, University of Oxford, UK.
Biochemistry. 1996 May 14;35(19):5982-91. doi: 10.1021/bi952756u.
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).
T细胞糖蛋白CD2是介导细胞表面识别的最具特征的分子之一。小鼠和人类CD2的配体分别是CD48和CD58，这些分子之间的相互作用已被证明会影响抗原识别和T细胞活化。人类CD2的CD58结合位点已在突变研究中得到表征，在此我们使用异核核磁共振光谱来鉴定大鼠CD2 N端结构域（CD2d1）的大鼠CD48结合位点。最初在确定该结构域的三维溶液结构过程中于pH 4.3下归属的细菌表达的CD2d1的核磁共振谱[德里斯科尔，P.C.等人（1991年）《自然》353，762 - 765]，已重新归属为中性pH下的二维15N - 1H异核单量子相干（HSQC）谱。通过监测在用中国仓鼠卵巢细胞中表达的可溶性形式的CD48滴定过程中CD2d1的HSQC谱中的交叉峰的线宽和化学位移的扰动来鉴定CD48结合表面。这种对相互作用的细胞表面分子的首次溶液核磁共振分析表明，配体结合位点跨越GFCC'C"面约700 - 800 Å2的区域，几乎与最初提出作为CD2与其配体相互作用模型的可溶性CD2晶体中的晶格接触完全对应。分析未发现CD2结构域1伴随CD48结合有任何大规模结构变化的证据。人类和大鼠CD2配体结合位点的比较表明，物种和配体特异性结合可能由少至三个氨基酸残基决定，对应于大鼠CD2中的Thr37、Leu38和Glu41（人类CD2中的Lys42、Lys43和Gln46）。
