title: Post-translational modification of ribosomal proteins: structural and functional characterization of RimO from Thermotoga maritima, a radical S-adenosylmethionine methylthiotransferase.
abstract: Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 A crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.
authors.affiliation: Institut de Recherches en Technologie et Sciences pour le Vivant-Laboratoire de Chimie et Biologie des Métaux (iRTSV-LCBM), UMR 5249, CEA-CNRS-UJF, Commissariat à l'Energie Atomique Grenoble, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Arragain
authors.forename: Ricardo
authors.initials: R
authors.lastname: Garcia-Serres
authors.forename: Geneviève
authors.initials: G
authors.lastname: Blondin
authors.forename: Thierry
authors.initials: T
authors.lastname: Douki
authors.forename: Martin
authors.initials: M
authors.lastname: Clemancey
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Latour
authors.forename: Farhad
authors.initials: F
authors.lastname: Forouhar
authors.forename: Helen
authors.initials: H
authors.lastname: Neely
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Etienne
authors.initials: E
authors.lastname: Mulliez
authors.forename: Marc
authors.initials: M
authors.lastname: Fontecave
authors.forename: Mohamed
authors.initials: M
authors.lastname: Atta
mesh_terms: D018360:Crystallography, X-Ray; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D012269:Ribosomal Proteins; D012436:S-Adenosylmethionine; D013466:Sulfurtransferases