title: The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement.
abstract: Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His(15), His(122) and Gln(14)) and cationic surface-exposed residues (His(36), His(39), Arg(66) and His(67)). Kinetic characterization, together with prediction of protein-nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5' end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His(36)-His(39) dyad that facilitates the polynucleotide substrate catalysis.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: Guillem
authors.initials: G
authors.lastname: Prats-Ejarque
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Arranz-Trullén
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Blanco
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pulido
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: M Victòria
authors.initials: MV
authors.lastname: Nogués
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moussaoui
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Vallès, Spain Ester.Boix@uab.cat.
authors.forename: Ester
authors.initials: E
authors.lastname: Boix
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D012259:Ribonuclease, Pancreatic
keywords: RNase A superfamily; RNase k6; kinetic characterization; molecular dynamics; protein crystallography; sulfate anion