Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Autor: Zhdanova PV; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia.; Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia., Chernonosov AA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia., Prokhorova DV; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia., Stepanov GA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia., Kanazhevskaya LY; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia., Koval VV; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia.; Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2022 Jan 20; Vol. 23 (3). Date of Electronic Publication: 2022 Jan 20.
DOI: 10.3390/ijms23031129
Abstrakt: The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.
Databáze: MEDLINE
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