Li Xintian, Lee Hunjoong, Wu Jin, Breslow Esther
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
Protein Sci. 2007 Jan;16(1):52-68. doi: 10.1110/ps.062444807.
Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.
目前的证据表明，神经垂体激素运载蛋白的配体促进二聚化部分是由未结合配体状态下激素结合位点的二聚化诱导变化介导的，这些变化增加了配体亲和力。为了阐明其他促成因素，我们研究了神经垂体激素运载蛋白短结构域间环（第55 - 59位氨基酸残基）的潜在作用，特别是环残基突变以及缺失与该环及相邻残基53 - 54相互作用的氨基末端残基1 - 6的影响。所研究的神经垂体激素运载蛋白是牛神经垂体激素运载蛋白-I，因此需要确定去1 - 6牛神经垂体激素运载蛋白-I在未结合配体和结合配体的二聚体状态下的晶体结构，以及其结合配体的Q58V突变体的结构，在该突变体中肽的结合亲和力意外增加。与选定的环残基突变以及残基1 - 6缺失相关的二聚化常数增加，连同结构数据，提供了证据表明未结合配体的神经垂体激素运载蛋白-I的二聚化受到Gln58、Ser56和Gln55侧链的氢键以及氨基末端相互作用的限制，这些氢键的丧失或改变以及氨基末端相互作用的可能丧失，促成了结合配体状态下二聚化的增加。仅在结合配体状态下存在的来自残基81形成的额外亚基间氢键被证明是配体直接作用于亚基界面的最大单一效应。牛神经垂体激素运载蛋白I和II的比较表明两者的机制大致相似，除了神经垂体激素运载蛋白II在未结合配体状态下不存在Gln第五十五侧链氢键，以及在结合配体状态下氨基末端相互作用的丧失更确定。有证据表明，环的状态通过对涉及相邻Phe22作为关键中介的神经垂体激素运载蛋白构象的远程影响来调节二聚化。
