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Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14.

ABSTRACT: Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9-sgRNA-dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.

SUBMITTER: Liu H

PROVIDER: S-EPMC8216286 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

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Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14.

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