title: The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases.
abstract: Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 A: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA(Pro) with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA(Pro) and tRNA(Cys) with their cognate amino acids.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
authors.forename: Satwik
authors.initials: S
authors.lastname: Kamtekar
authors.forename: W Dexter
authors.initials: WD
authors.lastname: Kennedy
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
authors.forename: Constantinos
authors.initials: C
authors.lastname: Stathopoulos
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Steitz
mesh_terms: D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D019605:Euryarchaeota / Q000201:enzymology; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012361:RNA, Transfer, Pro / Q000378:metabolism*
chemical_list: D012361:RNA, Transfer, Pro; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; D003545:Cysteine