Abstract
HIV-1 integrase (HIV-1 IN) is an enzyme produced by the HIV-1 virus that integrates genetic material of the virus into the DNA of infected human cells. HIV-1 IN acts as a key component of the Retroviral Pre-Integration Complex (PIC). Protein dynamics could play an important role during the catalysis of HIV-1 IN; however, this process has not yet been fully elucidated. X-ray free electron laser (XFEL) together with nuclear magnetic resonance (NMR) could provide information regarding the dynamics during this catalysis reaction. Here, we report the non-cryogenic crystal structure of HIV-1 IN catalytic core domain at 2.5 Å using microcrystals in XFELs. Compared to the cryogenic structure at 2.1 Å using conventional synchrotron crystallography, there was a good agreement between the two structures, except for a catalytic triad formed by Asp64, Asp116, and Glu152 (DDE) and the lens epithelium-derived growth factor binding sites. The helix III region of the 140–153 residues near the active site and the DDE triad show a higher dynamic profile in the non-cryogenic structure, which is comparable to dynamics data obtained from NMR spectroscopy in solution state.
Highlights
The study of protein dynamics has progressively improved with the discovery of experimental techniques such as hydrogen deuterium exchange mass spectrometry, molecular dynamics (MD) simulation, and solution nuclear magnetic resonance (NMR)
Both the cryogenic and non-cryogenic structures of IN catalytic core domain (IN-CCD) were solved by conventional synchrotron and SFX-X-ray free electron laser (XFEL) (Table 1)
In non-cryogenic structure from SFX-XFELs, there is no sign of radiation damage, such that the reduction in electron density caused by a diffuse electron density appears as a peak in the difference Fourier electron density map
Summary
The study of protein dynamics has progressively improved with the discovery of experimental techniques such as hydrogen deuterium exchange mass spectrometry, molecular dynamics (MD) simulation, and solution nuclear magnetic resonance (NMR). The methods that allow for the analysis of the structure and the dynamics of the protein simultaneously are limited to MD simulation and solution NMR. These techniques have limitations such as the size of molecules or the experimental support needed to validate the simulation results. Mix-and-inject serial crystallography (MISC) was designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate [3,4,5]. MISC is difficult, which limits its use in experiments
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