title: NMR-based substrate analog docking to Escherichia coli peptidyl-tRNA hydrolase.
abstract: Escherichia coli peptidyl-tRNA hydrolase activity is inhibited by 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, a stable mimic of the minimalist substrate 2'(3')-O-(L-[N,N-diacetyl-lysinyl)adenosine. The complex of this mimic with the enzyme has been analyzed by NMR spectroscopy, enabling experimental mapping of the catalytic center for the first time. Chemical shift variations point out the sensitivity of residues Asn10, Met67, Asn68, Gly111, Asn114, Leu116, Lys117, Gly147, Phe148, and Val149 to complex formation. Docking simulations based on ambiguous interaction restraints involving these residues show bondings of the peptide moiety of 3'-(l-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine with Asn10, Asn68, and Asn114. A stacking interaction of Phe66 with the purine is also indicated. Drawn is a model of enzyme-bound peptidyl-tRNA substrate, in which: (i) the Asn114 δ(2) NH(2) group holds the water molecule that participates in the hydrolysis of the substrate, while Tyr15 binds the phosphate in the 5'-position of the 3'-terminal tRNA adenosine; (ii) the δ(2) NH(2) group of Asn68 holds the main-chain carbonyl of the C-terminal residue of the peptide esterified to tRNA; and (iii) the δ(2) NH(2) group of Asn10 holds the main-chain carbonyl of the penultimate C-residue. Functional value is given to this model by (i) showing that the enzyme becomes confusable with an aminoacyl-tRNA hydrolase upon mutagenesis of Asn10 and (ii) reinterpreting already obtained site-directed mutagenesis data.
authors: Laboratoire de Biochimie, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
authors: Laurent
authors: L
authors: Giorgi
authors: Pierre
authors: P
authors: Plateau
authors: Gavin
authors: G
authors: O'Mahony
authors: Caroline
authors: C
authors: Aubard
authors: Michel
authors: M
authors: Fromant
authors: Aurélien
authors: A
authors: Thureau
authors: Morten
authors: M
authors: Grøtli
authors: Sylvain
authors: S
authors: Blanquet
authors: François
authors: F
authors: Bontems
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase