RESUMEN
The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.
Asunto(s)
Calcio/metabolismo , Canales de Cloruro/química , Canales de Cloruro/metabolismo , Nectria/química , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Animales , Anoctamina-1 , Sitios de Unión/genética , Calcio/química , Calcio/farmacología , Canales de Cloruro/genética , Cristalografía por Rayos X , Conductividad Eléctrica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Transporte Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nectria/enzimología , Nectria/genética , Proteínas de Neoplasias/química , Proteínas de Transferencia de Fosfolípidos/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.