Kim, Iktae Jeong, Migyeong Ka, Donghyun Han, Mookyoung Kim, Nak-Kyoon Bae, Euiyoung Suh, Jeong-Yong 2024-01-19T23:04:24Z 2024-01-19T23:04:24Z 2021-09-03 2018-03-01 2045-2322 https://pubs.kist.re.kr/handle/201004/121620 The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric alpha beta beta beta alpha alpha fold comprising three antiparallel beta strands flanked by three alpha-helices and a short 3(10)-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the beta 1-beta 2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the beta 1-beta 2 and alpha 2-alpha 3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with K-D similar to 4.8 mu M, which compares to K-D similar to 0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA. English NATURE PUBLISHING GROUP STRUCTURE REFINEMENT DIPOLAR COUPLINGS DISTANCE GEOMETRY NMR-SPECTROSCOPY CHEMICAL-SHIFTS SYSTEMS COMPLEXES VISUALIZATION PROKARYOTES PROTEINS Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor Article 10.1038/s41598-018-22177-0 1 SCIENTIFIC REPORTS, v.8 SCIENTIFIC REPORTS 8 scie scopus 000426354200055 2-s2.0-85042766811 Multidisciplinary Sciences Science & Technology - Other Topics Article STRUCTURE REFINEMENT DIPOLAR COUPLINGS DISTANCE GEOMETRY NMR-SPECTROSCOPY CHEMICAL-SHIFTS SYSTEMS COMPLEXES VISUALIZATION PROKARYOTES PROTEINS CRISPR Cas9 inhibitor AcrIIA4 structure dynamics