RESUMO
The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.
Assuntos
Sistemas CRISPR-Cas , Microscopia Crioeletrônica/métodos , DNA/química , RNA Guia de Cinetoplastídeos , RNA/química , Motivos de Aminoácidos , Cianobactérias , DNA/metabolismo , Edição de Genes , Técnicas Genéticas , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Domínios Proteicos , Recombinação GenéticaRESUMO
Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.
Assuntos
Bacteriófagos , Proteínas Associadas a CRISPR , Proteínas Associadas a CRISPR/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sistemas CRISPR-Cas , Bacteriófagos/genética , Bacteriófagos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
This study aimed to identify the intracellular binding partner of a unique class of staphylococcal secreted exotoxins called superantigen-like proteins (SSL) from human macrophage and keratinocyte cell lysates. Here, we report that SSL1 specifically binds to human extracellular signal-regulated kinase 2 (hERK2), an important stress-activated kinase in mitogen-activated protein kinase signaling pathways. Western blot and in vitro binding studies with recombinant hERK2 confirmed the binding interaction of SSL1, SSL7, and SSL10 with hERK2. Moreover, the SSLs-hERK2 interaction was validated biochemically by ELISA. Our finding shows that SSLs play a novel role by binding with host cell MAP kinase signaling pathway protein. Understanding the SSL-hERK2 interaction will also provide a basis for designing SSL-based peptide inhibitors of hERK2 in cancer therapy.