Structures of Bacterial Polynucleotide Kinase in a Michaelis Complex with Nucleoside Triphosphate (NTP)-Mg 2+ and 5′-OH RNA and a Mixed Substrate-Product Complex with NTP-Mg 2+ and a 5′-Phosphorylated Oligonucleotide

Abstract

ABSTRACT Clostridium thermocellum polynucleotide kinase ( Cth Pnk), the 5′-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5′-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of Cth Pnk-D38N in a Michaelis complex with GTP-Mg 2+ and a 5′-OH RNA oligonucleotide. The RNA-binding mode of Cth Pnk is different from that of the metazoan RNA kinase Clp1. Cth Pnk makes hydrogen bonds to the ribose 2′-hydroxyls of the 5′ terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5′-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active Cth Pnk with GTP-Mg 2+ and a 5′-OH DNA yielded a mixed substrate-product complex with GTP-Mg 2+ and 5′-PO 4 DNA, wherein the product 5′ phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.