Serial Femtosecond X-Ray Diffraction of HIV-1 Gag MA-IP6 Microcrystals at Ambient Temperature.

Halil I Ciftci,Hasan Demirci,Toshiya Senda,Mikako Fujita,Zhen Su,Hiroshi Tateishi,Koiwai Kotaro,Chun Yoon,Mengling Liang,Soichi Wakatsuki,Masami Otsuka,Raymond G Sierra,Ryoko Koga,Fumiaki Yumoto

doi:10.3390/ijms20071675

Abstract

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.

Highlights

Based on the structural information of the MA domain and PI(4,5)P2, we recently developed a non-natural derivative of PI(4,5)P2, named L-HIPPO, which binds to the MA domain in order to eradicate human immunodeficiency virus (HIV) [20,21]
We describe the experimental procedures from purification and characterization of the Human immunodeficiency virus-1 (HIV-1) MA protein, its large-scale co-crystallization with IP6 in two crystal forms, and efficient delivery of these crystals for ambient-temperature diffraction data collection through an serial femtosecond X-ray crystallography (SFX) experiment
Diffraction patterns of MA-IP6 microcrystals were recorded beyond 3.5 Å resolution

Summary

Methods

Cloning and Overexpression of the HIV-1 Gag MA Domain. Cells were harvested in lysis buffer containing 20 mM 2-Amino-2-(hydroxymethyl) propane-1,3-diol (Tris)-HCl pH 8.0, 0.5 M NaCl, 30 mM imidazole, 5 mM β-mercaptoethanol and lysed by sonication. The remaining supernatants, which contain the soluble MA fraction, were pooled and loaded onto a Nickel-Nitriloacetic acid (Ni-NTA) column and washed with 10 times column volume of wash buffer containing 20 mM Tris-HCl pH 8.0, 1 M NaCl, 1 M (NH4)2SO4, 30 mM imidazole and 5 mM β-mercaptoethanol. Bound MA fractions were eluted with elution buffer containing 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 300 mM imidazole and 5 mM β-mercaptoethanol, and buffer exchanged, and the His10-tag was cleaved off with Tobacco Etch Virus (TEV) protease at pH 8.0.

Results

Discussion

Conclusion