title: "Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease, RNase E.
abstract: Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
authors.forename: Anastasia J
authors.initials: AJ
authors.lastname: Callaghan
authors.forename: Yulia
authors.initials: Y
authors.lastname: Redko
authors.forename: Loretta M
authors.initials: LM
authors.lastname: Murphy
authors.forename: J Günter
authors.initials: JG
authors.lastname: Grossmann
authors.forename: David
authors.initials: D
authors.lastname: Yates
authors.forename: Elspeth
authors.initials: E
authors.lastname: Garman
authors.forename: Leopold L
authors.initials: LL
authors.lastname: Ilag
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Martyn F
authors.initials: MF
authors.lastname: Symmons
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: McDowall
authors.forename: Ben F
authors.initials: BF
authors.lastname: Luisi
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003958:Diamide / Q000737:chemistry; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013052:Spectrometry, X-Ray Emission; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013499:Surface Properties; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D013439:Sulfhydryl Reagents; D003958:Diamide; D004722:Endoribonucleases; C022537:ribonuclease E; D015032:Zinc