Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system Journal Article


Authors: Wang, L. K.; Das, U.; Smith, P.; Shuman, S.
Article 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 a and b 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. Copyright © 2012 RNA Society.
Keywords: unclassified drug; nonhuman; amino acid substitution; serine; carboxy terminal sequence; bacterial protein; bacterial proteins; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; protein multimerization; recombinant proteins; binding site; crystal structure; hydrogen bond; models, molecular; dimerization; crystallography, x-ray; mutagenesis, site-directed; protein structure, tertiary; threonine; catalysis; protein folding; protein secondary structure; protein structure; crystallization; catalytic domain; protein tertiary structure; lysine; polynucleotide 5' hydroxyl kinase; polynucleotide 5'-hydroxyl-kinase; arginine; enzyme active site; rna, bacterial; polynucleotide; rna end-healing; rna repair; clostridium thermocellum; helix loop helix protein; homodimer; beta sheet; p-loop phosphotransferase; adenosine diphosphate magnesium; protein hen 1; protein pnkp
Journal Title: RNA
Volume: 18
Issue: 12
ISSN: 1355-8382
Publisher: Cold Spring Harbor Laboratory Press  
Date Published: 2012-12-01
Start Page: 2277
End Page: 2286
Language: English
DOI: 10.1261/rna.036061.112
PROVIDER: scopus
PMCID: PMC3504678
PUBMED: 23118415
DOI/URL:
Notes: --- - "Export Date: 2 January 2013" - "CODEN: RNARF" - "Source: Scopus"
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MSK Authors
  1. Li-Kai Wang
    27 Wang
  2. Stewart H Shuman
    546 Shuman
  3. Paul M C Smith
    21 Smith
  4. Ushati Das
    11 Das