Doležal Michal, Zábranský Aleš, Dostál Jiří, Vaněk Ondřej, Brynda Jiří, Lepšík Martin, Hadravová Romana, Pichová Iva
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10, Prague, Czech Republic.
Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 40, Prague, Czech Republic.
Retrovirology. 2016 Jan 5;13:2. doi: 10.1186/s12977-015-0235-8.
Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated.
Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release.
Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.
基质（MA）结构域的肉豆蔻酰化介导了 gag 多聚蛋白向质膜（PM）的转运和结合，是大多数逆转录病毒组装所必需的。在β逆转录病毒中，其在细胞质中组装不成熟颗粒，肉豆蔻酰化对于组装不是必需的，但对于颗粒向质膜的转运至关重要。HIV-1 MA 的寡聚化刺激肉豆蔻酰基从隔离构象转变为暴露构象，从而更易于与膜结合。然而，对于其他逆转录病毒，MA 寡聚化对肉豆蔻酰基暴露的影响尚未得到充分研究。
在这里，我们证明来自β逆转录病毒小鼠乳腺肿瘤病毒（MMTV）的 MA 在溶液中形成二聚体，并且这个过程受到其肉豆蔻酰化的刺激。以 1.57 Å 分辨率测定的 N-肉豆蔻酰化 MMTV MA 的晶体结构表明，肉豆蔻酰基埋在二聚体界面的疏水口袋中，并有助于二聚体形成。有趣的是，如分子动力学建模所示，二聚体中的肉豆蔻酰基相互交换以实现能量稳定的结合。肉豆蔻酰结合位点内的突变导致 MA 二聚化减少和细胞外颗粒释放减少。
基于我们的实验、结构和计算数据，我们提出了一个 MMTV MA 二聚化的模型，其中肉豆蔻酰基刺激 MA 分子之间的相互作用。此外，形成二聚体的 MA 分子采用隔离构象，其肉豆蔻酰基完全埋在相互作用界面内。尽管这与目前为慢病毒提出的模型不同，在慢病毒模型中 MA 的寡聚化触发肉豆蔻酰基的暴露，但这似乎便于细胞内组装，因为细胞内组装不涉及明显的膜相互作用，并且允许肉豆蔻酰基在寡聚化过程中被隔离。
