title: Enhanced amino acid selection in fully evolved tryptophanyl-tRNA synthetase, relative to its urzyme, requires domain motion sensed by the D1 switch, a remote dynamic packing motif.
abstract: We previously showed (Li, L., and Carter, C. W., Jr. (2013) J. Biol. Chem. 288, 34736-34745) that increased specificity for tryptophan versus tyrosine by contemporary Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) over that of TrpRS Urzyme results entirely from coupling between the anticodon-binding domain and an insertion into the Rossmann-fold known as Connecting Peptide 1. We show that this effect is closely related to a long range catalytic effect, in which side chain repacking in a region called the D1 Switch, accounts fully for the entire catalytic contribution of the catalytic Mg(2+) ion. We report intrinsic and higher order interaction effects on the specificity ratio, (kcat/Km)Trp/(kcat/Km)Tyr, of 15 combinatorial mutants from a previous study (Weinreb, V., Li, L., and Carter, C. W., Jr. (2012) Structure 20, 128-138) of the catalytic role of the D1 Switch. Unexpectedly, the same four-way interaction both activates catalytic assist by Mg(2+) ion and contributes -4.4 kcal/mol to the free energy of the specificity ratio. A minimum action path computed for the induced-fit and catalytic conformation changes shows that repacking of the four residues precedes a decrease in the volume of the tryptophan-binding pocket. We suggest that previous efforts to alter amino acid specificities of TrpRS and glutaminyl-tRNA synthetase (GlnRS) by mutagenesis without extensive, modular substitution failed because mutations were incompatible with interdomain motions required for catalysis.
authors.affiliation: From the Department of Biochemistry and Biophysics, CB 7260, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260.
authors.forename: Violetta
authors.initials: V
authors.lastname: Weinreb
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Srinivas Niranj
authors.initials: SN
authors.lastname: Chandrasekaran
authors.forename: Patrice
authors.initials: P
authors.lastname: Koehl
authors.forename: Marc
authors.initials: M
authors.lastname: Delarue
authors.forename: Charles W
authors.initials: CW
authors.lastname: Carter
mesh_terms: D020816:Amino Acid Motifs; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase; D014369:Tryptophan-tRNA Ligase
keywords: Allostery; Aminoacyl tRNA Synthesis; Conformational Change; Cooperativity; Enzyme Mechanisms; Minimum Action Path; Mutagenesis Site Specific; Pocket Volume; Thermodynamics; Transition State