Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity
Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity
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DOI：
10.1006/jmbi.1998.1669
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发表时间：
1998-04-17
影响因子：
5.6
通讯作者：
de Vos, AM
中科院分区：
文献类型：
--
作者：
Clackson, T;Ultsch, MH;de Vos, AM
设计的人生长激素（hGH）的G120 R突变体是拮抗剂，并且只能结合生长激素受体的一个分子。我们已经确定了该突变体和受体胞外结构域（hGHbp）之间的1：1复合物的晶体结构，分辨率为2.6埃，并用它来指导详细调查的结构和功能的基础，为hGHbp受体识别。复合物的整体结构与1：2复合物的等效部分非常相似，表明活性复合物的形成不涉及主要构象变化。然而，在1：2复合物中参与受体-受体相互作用的片段在该结构中是无序的，这表明其生产性构象通过受体二聚化而稳定，受体的激素结合位点包括由Trp 104和Trp 169主导的中央疏水补丁，周围是含有几个有序水分子的亲水外围。先前的丙氨酸扫描显示疏水性“热点”赋予大部分结合能。新的结构数据，再加上进一步的突变体的结合和动力学分析，表明热点组装合作，许多残基有助于间接结合。几个疏水残基用于定位关键的色氨酸残基;动力学分析表明，Pro 106缺乏Trp 104主链进入所需的构象。Arg 43与hGH的静电接触不如其烷基链与Trp 169的分子内堆积重要。因此，直接贡献结合能的真正功能表位可以包含少至六个侧链，主要参与烷基-芳族堆积相互作用。在功能表位之外，残基向丙氨酸的多个突变导致亲和力的非加性增加：对于七丙氨酸突变体高达十倍。因此，表位外围的接触可以减弱中心热点的亲和力，这可能反映了赋予相互作用特异性的作用。(C)出版社：Academic Press Limited。
The designed G120R mutant of human growth hormone (hGH) is an antagonist and can bind only one molecule of the growth hormone receptor. We have determined the crystal structure of the 1:1 complex between this mutant and the receptor extracellular domain (hGHbp) at 2.6 Angstrom resolution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall structure of the complex is very similar to the equivalent portion of the 1:2 complex, showing that formation of the active complex does not involve major conformational changes. However, a segment involved in receptor-receptor interactions in the 1:2 complex is disordered in this structure, suggesting that its productive conformation is stabilized by receptor dimerization.The hormone binding site of the receptor comprises a central hydrophobic patch dominated by Trp104 and Trp169, surrounded by a hydrophilic periphery containing several well-ordered water molecules. Previous alanine scanning showed that the hydrophobic "hot spot" confers most of the binding energy. The new structural data, coupled with binding and kinetic analysis of further mutants, indicate that the hot spot is assembled cooperatively and that many residues contribute indirectly to binding. Several hydrophobic residues serve to orient the key tryptophan residues; kinetic analysis suggests that Pro106 lacks the Trp104 main-chain into a required conformation. The electrostatic contacts of Arg43 to hGH are less important than the intramolecular packing of its alkyl chain with Trp169. The true functional epitope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacking interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periphery can therefore attenuate the affinity of the central hot spot, perhaps reflecting a role in conferring specificity to the interaction. (C) 1998 Academic Press Limited.