A combined molecular dynamics and rapid kinetics approach to identify conserved three-dimensional communication networks in elongation factor Tu.
Elongation factor (EF) Tu delivers aminoacyl-tRNAs to the actively translating bacterial ribosome in a GTP-hydrolysis-dependent process. Rapid recycling of EF-Tu, catalyzed by EF-Ts, is required for efficient protein synthesis in vivo. Here we report a combined theoretical and experimental approach aimed at identifying three-dimensional communication networks in EF-Tu. As an example, we focus on the mechanistic role of second-shell residue Asp(109). We constructed full-length structural models of EF-Tu from Escherichia coli in the GDP-/GTP-bound state and performed several 10-ns-long molecular-dynamics simulations. During these simulations, the side chain of Asp(109) formed a previously undetected transient hydrogen bond to His(22), an invariant residue in the phosphate-binding loop (P-loop). To experimentally validate our molecular-dynamics results and further analyze the role of this hydrogen bond, we determined all rate constants for the multistep reaction between EF-Tu (wild-type and two mutants), EF-Ts, GDP, and GTP using the stopped-flow technique. This mutational analysis revealed that the side chain of Asp(109) is important for acceleration of GDP, but not for GTP dissociation by EF-Ts. The possibility that the Asp(109) side chain has a role in transition-state stabilization and coupling of P-loop movements with rearrangements at the base side of the nucleotide is discussed.
Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada. hj.wieden@uleth.ca
Hans-Joachim
HJ
Wieden
Evan
E
Mercier
John
J
Gray
Brett
B
Steed
Davis
D
Yawney
D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D017124:Conserved Sequence / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
D000596:Amino Acids; D050505:Mutant Proteins; D010445:Peptide Elongation Factors; C023512:elongation factor Ts; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu