title: A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu.
abstract: The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu · GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.
authors.forename: Evan
authors.initials: E
authors.lastname: Mercier
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.
authors.forename: Dylan
authors.initials: D
authors.lastname: Girodat
authors.affiliation: Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Wieden
mesh_terms: D001665:Binding Sites; D019277:Entropy; D004926:Escherichia coli / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu