title: Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system.
abstract: Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5'-kinase, a central 2',3' phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATP•Mg²⁺ (substrate complex) and ADP•Mg²⁺ (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. ATP is bound within a crescent-shaped groove formed by the P-loop (¹⁵GSSGSGKST²³) and an overlying helix-loop-helix "lid." The α and β phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the γ phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg²⁺ bridge the ATP β and γ phosphates. The P-loop serine (Ser22) is the sole enzymic constituent of the octahedral metal coordination complex. Structure-guided mutational analysis underscored the essential contributions of Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 5'-OH for its nucleophilic attack on the γ phosphorus and Lys21 and Mg²⁺ stabilize the transition state.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Li Kai
authors.initials: LK
authors.lastname: Wang
authors.forename: Ushati
authors.initials: U
authors.lastname: Das
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D011116:Polynucleotide 5'-Hydroxyl-Kinase