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:	http://www.scopus.com/inward/record.url?eid=2-s2.0-84869782421&partnerID=40&md5=47a352a64895bc487194a1929bcf7344
Notes:	--- - "Export Date: 2 January 2013" - "CODEN: RNARF" - "Source: Scopus"

https://synapse.mskcc.org/synapse/works/has_related_data_chk/37437