Department of Drug Discovery, Science Farm Ltd., Kumamoto 862-0976, Japan.
Department of Bioorganic Medicinal Chemistry, School of Pharmacy, Kumamoto University, Kumamoto 862-0973, Japan.
Int J Mol Sci. 2019 Apr 3;20(7):1675. doi: 10.3390/ijms20071675.
The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cell's plasma membrane. High-resolution structures of the MA domain determined using cryo X-ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first X-ray free-electron laser (XFEL) study of the HIV-1 MA domain's interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using an XFEL to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of the MA-IP6 complex both diffracted to beyond 3.5 Å resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and will provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.
人类免疫缺陷病毒 1（HIV-1）基质（MA）结构域参与病毒颗粒在宿主细胞质膜处的高度调控组装过程。使用冷冻 X 射线晶体学确定的 MA 结构域的高分辨率结构为病毒组装过程中的可能步骤提供了初步的见解。然而，这些结构研究依赖于大的和冷冻的晶体，以减少强烈 X 射线引起的辐射损伤。在这里，我们报告了使用 X 射线自由电子激光（XFEL）首次研究 HIV-1 MA 结构域与肌醇六磷酸（IP6）的相互作用，IP6 是一种磷脂头部基团模拟物。我们还描述了在存在 IP6 的情况下获得的两种 MA 晶体形式的纯化、表征和微结晶。此外，我们描述了使用 XFEL 进行串行 femtosecond X 射线晶体学（SFX）的能力，以阐明在环境温度下在液体悬浮液中 MA-IP6 配合物微晶体的衍射数据。两种不同形式的 MA-IP6 配合物微晶体都能衍射到 3.5Å分辨率以上，证明了使用 SFX 研究 HIV-1 Gag 多蛋白的 MA 结构域与 IP6 之间在接近生理温度下的复合物的可行性。进一步优化实验和数据分析程序将有助于更好地理解 HIV-1 Gag 的 MA 结构域和 IP6 相互作用的高分辨率，并为优化先导化合物以有效抑制 Gag 蛋白在形成病毒颗粒之前募集到质膜提供基础。
