title: Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases.
abstract: Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.
authors.affiliation: Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, Wisconsin 54702, United States.
authors.forename: Brianne
authors.initials: B
authors.lastname: Sanford
authors.forename: Bach
authors.initials: B
authors.lastname: Cao
authors.forename: James M
authors.initials: JM
authors.lastname: Johnson
authors.forename: Kurt
authors.initials: K
authors.lastname: Zimmerman
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Strom
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Mueller
authors.forename: Sudeep
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
authors.forename: Sanchita
authors.initials: S
authors.lastname: Hati
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D004269:DNA, Bacterial / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012361:RNA, Transfer, Pro / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017385:Sequence Homology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012361:RNA, Transfer, Pro; D011994:Recombinant Proteins; D011392:Proline; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase