RESUMEN
DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5'-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5'-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn(2+) complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove-binding C(2)HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5'-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5'-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).
Asunto(s)
Apraxias/genética , Apraxias/fisiopatología , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/fisiopatología , Proteínas de Unión al ADN/química , ADN/química , Hipoalbuminemia/genética , Hipoalbuminemia/fisiopatología , Proteínas Nucleares/química , Secuencias de Aminoácidos , Sitios de Unión , Ataxia Cerebelosa/congénito , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Roturas del ADN de Cadena Simple , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Dedos de ZincRESUMEN
EXTL2, an alpha1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.
Asunto(s)
Heparitina Sulfato/biosíntesis , Proteínas de la Membrana , N-Acetilglucosaminiltransferasas/química , Secuencia de Aminoácidos , Animales , Arginina/química , Asparagina/química , Ácido Aspártico/química , Sitios de Unión , Células COS , Dominio Catalítico , Cristalografía por Rayos X , Heparitina Sulfato/química , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , N-Acetilgalactosaminiltransferasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transfección , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 A in a ternary complex with 3'-phosphoadenosine 5'-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.