Department of Microbiology, University of Alabama, Birmingham, Alabama 35294, USA.
J Biol Chem. 2010 Dec 31;285(53):41911-20. doi: 10.1074/jbc.M110.179093. Epub 2010 Oct 18.
Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca(2+)-binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(+)MA) domain. Herein, we demonstrate that CaM binds to myr(+)MA with a dissociation constant (K(d)) of ∼2 μm and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(+)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.
在确定逆转录病毒组装和释放的病毒和细胞决定因素方面已经取得了稳步进展。尽管人们普遍认为，Gag 多肽靶向质膜对于 HIV-1 的正确组装至关重要，但 Gag 在内质网中的相互作用和运输及其在感染细胞中的组装部位仍知之甚少。研究表明，HIV-1 Gag 与钙调蛋白（CaM）相互作用并共定位，钙调蛋白是一种广泛存在且高度保守的 Ca（2+）结合蛋白，存在于所有真核细胞中，并参与多种细胞功能。HIV-1 Gag 与 CaM 的结合依赖于钙，并且由 N 端豆蔻酰化基质（myr(+)MA）结构域介导。在此，我们证明 CaM 与 myr(+)MA 的结合解离常数（K(d)）为∼2 μm，且具有 1:1 的化学计量比。引人注目的是，我们的数据显示，CaM 与 MA 的结合会导致豆蔻酰基团的挤出。然而，与所有已知的 CaM 结合豆蔻酰化蛋白的例子不同，我们的数据表明，豆蔻酰基团暴露在溶剂中，不参与 CaM 结合。CaM 和 myr(+)MA 之间的相互作用是吸热的并且由熵驱动，这表明疏水相互作用对于结合至关重要。如 NMR 数据所示，CaM 和 MA 似乎都在结合时涉及到大量区域和/或发生显著的构象变化。我们相信，我们的发现将为 Gag 在 HIV 复制周期中如何与 CaM 相互作用提供新的见解。
