The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR
The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR
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DOI：
10.1021/bi9611164
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发表时间：
1996-08-13
期刊：
影响因子：
2.9
通讯作者：
Bax, A
中科院分区：
文献类型：
--
作者：
Grzesiek, S;Stahl, SJ;Bax, A
使用异源NMR光谱，我们证明，13个残基的肽（MSQIKRLLSEKKT）从CD 4的细胞质尾结合Nef蛋白。CD 4的这一部分对于HIV-1 Nef下调CD 4至关重要[Aiken et al.（1994）Cell 76，853-864]。我们表明，没有中央双亮氨酸的对照肽不结合Nef。Nef H-1和N-15酰胺化学位移对肽浓度的依赖性表明，结合处于快速化学交换极限，解离常数K-d类似于1 mM。肽结合位点已被映射到先前确定的HIV-1 Nef的溶液结构上[Grzesiek等（1996）Nat.Struct.Biol.3，340-345]基于肽诱导的化学位移变化。其包含氨基酸W57、L58、E59、G95、G96、L97、R106和L110。当Nef与Hck酪氨酸蛋白激酶的SH 3结构域复合时，肽结合Nef上的相同位点，但亲和力略高（K-d类似于0.5 mM）。这表明，CD 4和Hck SH 3与Nef的结合是两个相容的和轻微合作的事件。
Using heteronuclear NMR spectroscopy, we demonstrate that a 13-residue peptide (MSQIKRLLSEKKT) from the cytoplasmic tail of CD4 binds to Nef protein. This part of CD4 is critical for downregulation of CD4 by HIV-1 Nef [Aiken el al. (1994) Cell 76, 853-864]. We show that a control peptide without the central dileucine does not bind to Nef. The dependence of Nef H-1 and N-15 amide chemical shifts on peptide concentration indicates that the binding is in the fast chemical exchange limit, with a dissociation constant K-d of similar to 1 mM, The peptide binding site has been mapped onto the previously determined solution structure of HIV-1 Nef [Grzesiek ct al. (1996) Nat. Struct. Biol. 3, 340-345] on the basis of peptide-induced chemical shift changes. It comprises amino acids W57, L58, E59, G95, G96, L97, R106, and L110. When Nef is complexed to the SH3 domain of Hck tyrosine protein kinase, the peptide binds to the same site on Nef but with slightly higher affinity (K-d similar to 0.5 mM). This indicates that the binding of CD4 and Hck SH3 to Nef are two compatible and slightly cooperative events.