Arold S, Franken P, Strub M P, Hoh F, Benichou S, Benarous R, Dumas C
Centre de Biochimie Structurale, UMR C9955 CNRS, U414 INSERM, Université Montpellier I, Faculté de Pharmacie, France.
Structure. 1997 Oct 15;5(10):1361-72. doi: 10.1016/s0969-2126(97)00286-4.
Human immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal transduction and host cell activation. Nef has been shown to bind specifically to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src kinase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS.
We have determined the crystal structures of the conserved core of HIV-1 Nef protein alone and in complex with the wild-type SH3 domain of the p59fyn protein tyrosine kinase (Fyn), at 3.0 A resolution. Comparison of the bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disordered in the absence of the binding partner; this motif only fully adopts a left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-called RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic pocket of Nef as the isoleucine residue of a previously described Fyn SH3 (Arg96-->lle) mutant that binds to Nef with higher affinity than the wild type.
The three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.
人类免疫缺陷病毒（HIV）Nef蛋白通过与参与信号转导和宿主细胞激活的特定细胞蛋白相互作用，加速获得性免疫缺陷综合征（AIDS）的恶性进展。已证明Nef能特异性结合Src激酶家族的一个亚群。游离Nef以及与Src同源区3（SH3）结构域结合的Nef的结构，对于阐明如何实现对Src激酶家族SH3结构域的亲和力和特异性很重要，也有助于开发抗艾滋病的潜在药物和疫苗。
我们已确定HIV-1 Nef蛋白保守核心单独以及与p59fyn蛋白酪氨酸激酶（Fyn）的野生型SH3结构域形成复合物时的晶体结构，分辨率为3.0埃。结合态和未结合态Nef结构的比较表明，与SH3结合有关的富含脯氨酸基序（Pro-x-x-Pro）在没有结合伴侣时部分无序；该基序只有在与Fyn SH3结构域形成复合物时才完全呈现左手多聚脯氨酸II型螺旋构象。此外，结构显示Nef的一个精氨酸残基（Arg77）如何与Fyn SH3结构域内所谓RT环的Asp 100相互作用，并引发氢键重排，使该环适应以补充Nef表面。Fyn SH3结构域的Arg96残基与先前描述的Fyn SH3（Arg96→Ile）突变体的异亮氨酸残基一样，特异性地容纳在Nef的同一疏水口袋中。该突变体与Nef的结合亲和力高于野生型。
三维结构支持以下证据：Nef-Fyn复合物在体内形成，并且可能通过改变T细胞受体信号传导在Nef的T细胞干扰作用中起关键作用。结合态和未结合态Nef的结构表明，SH3结合的多价性可能通过RT环中配体诱导的灵活性来实现。这些结构为设计特异性阻断Nef-SH3相互作用的抑制剂提供了可能的靶点。
