A mutational analysis of the binding of two different proteins to the same antibody
A mutational analysis of the binding of two different proteins to the same antibody
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DOI：
10.1021/bi960819i
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发表时间：
1996-07-30
期刊：
影响因子：
2.9
通讯作者：
Mariuzza, RA
中科院分区：
文献类型：
--
作者：
DallAcqua, W;Goldman, ER;Mariuzza, RA
抗鸡卵清溶菌酶（HEL）抗体D1.3与HEL以及D1.3与抗D1.3抗体E5.2之间复合物的晶体结构表明，D1.3通过基本相同的一组结合位点残基与这两种蛋白质相互作用[菲尔兹，B.A.，戈德鲍姆，F.A.，伊斯恩，X.，波利亚克，R.J.，&马里乌扎，R.A.（1995年）《自然》374，739 - 742]。为了探究单个残基对复合物稳定性的相对贡献，在D1.3的结合位点引入了单个丙氨酸替代，并使用表面等离子体共振检测、荧光猝灭滴定或沉降平衡测量了它们对HEL和E5.2亲和力的影响。与HEL结合的能量学仅由所测试的13个接触残基中的3个主导（ΔG（突变体） - ΔG（野生型）> 2.5千卡/摩尔）：V（L）W92、V（H）D100和V（H）Y101。这些在界面中心形成一个斑块，并被那些表观贡献不太显著（< 1.5千卡/摩尔）的残基所包围。这与D1.3和E5.2的相互作用形成对比，在D1.3与E5.2的相互作用中，大多数接触残基（15个中的11个）被发现在配体结合中起重要作用（> 1.5千卡/摩尔）。此外，尽管D1.3以非常相似的方式与HEL和E5.2相互作用，但对于这两种相互作用，D1.3的功能重要残基是不同的，只有在D1.3的V（H）100和V（H）101位置的替代对两种配体的结合都有很大影响。因此，同一种蛋白质可能以结构相似但能量不同的方式识别不同的配体。
The crystal structures of the complexes between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2 have shown that D1.3 contacts these two proteins through essentially the same set of combining site residues [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739-742], To probe the relative contribution of individual residues to complex stabilization, single alanine substitutions were introduced in the combining site of D1.3, and their effects on affinity for HEL and for E5.2 were measured using surface plasmon resonance detection, fluorescence quench titration, or sedimentation equilibrium, The energetics of the binding to HEL are dominated by only 3 of the 13 contact residues tested (Delta G(mutant) - Delta G(wild type) > 2.5 kcal/mol): V(L)W92, V(H)D1C0, and V(H)Y101. These form a patch at the center of the interface and are surrounded by residues whose apparent contributions are much less pronounced (< 1.5 kcal/mol). This contrasts with the interaction of D1.3 with E5.2 in which most the contact residues (11 of 15) were found to play a significant role in ligand binding (> 1.5 kcal/mol). Furthermore, even though D1.3 contacts HEL and E5.2 in very similar ways, the functionally important residues of D1.3 are different for the two interactions, with only substitutions at D1.3 positions V(H)100 and V(H)101 greatly affecting binding to both ligands. Thus, the same protein may recognize different ligands in ways that are structurally similar yet energetically distinct.