Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.
Proteins. 2011 May;79(5):1609-22. doi: 10.1002/prot.22986. Epub 2011 Mar 1.
Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.
Nef 是一种 HIV 辅助蛋白，在病毒感染后疾病的进展中起着重要作用。它干扰了许多信号通路，其中一条涉及丝氨酸/苏氨酸激酶。在这里，我们报告了全长 Nef 及其与 Hck 全长调节域（残基 72-256；Hck32L）相互作用的 NMR 结构研究结果。在折叠核心结构域之前，发现 N 端残基 14-22 处存在一个螺旋构象。与先前研究的截短 Nef（Nef Δ1-39）不同，全长 Nef 没有显示任何色氨酸 57/亮氨酸 58 与由 α1 和 α2 螺旋形成的疏水性斑之间的相互作用。在 Hck32L 结合后，N 端锚定结构域以及 Nef 的众所周知的 SH3 结合位点都表现出显著的化学位移变化。在 Nef 结合后，Hck 谱的共振变化主要局限于 SH3 结构域，在 SH3 和 SH2 之间的连接、SH2 的 N 端区域和包含调节多脯氨酸基序的连接区域也观察到了附加效应。结合数据表明，全长 Nef 中的核心结构域部分参与了相互作用。使用 RDC 数据对 Hck32L 的溶液构象进行建模，并与失活全长 Hck 中等效区域的晶体结构进行比较，揭示了 SH3 和 SH2 结构域的相对取向存在显著差异。基于 RDC 的模型结合（15）N 骨架动力学数据表明，Hck32L 采用开放构象，连接子中多脯氨酸基序不与 SH3 结构域结合。
