The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange.
Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.
From the Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
Evelina Ines
EI
De Laurentiis
From the Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
Evan
E
Mercier
From the Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada hj.wieden@uleth.ca.
Hans-Joachim
HJ
Wieden
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D009711:Nucleotides; D010445:Peptide Elongation Factors; C023512:elongation factor Ts; D010444:Peptide Elongation Factor Tu
GTPase; Pseudomonas aeruginosa (P. aeruginosa); catalytic mechanism; elongation factor Ts; elongation factor Tu; molecular dynamics; nucleotide binding; nucleotide exchange; pre-steady-state kinetics; translation elongation factor