A Conserved Arginine in Switch I Is Critical for ppGpp Binding to the Prokaryotic Translational GTPase BipA

Abstract

BipA is a highly conserved translational GTPase that functions in a variety of cellular processes including fitness, pathogenicity and acclimation to adverse growth conditions. Structural and biochemical studies indicate that GTP and ppGpp compete for binding to BipA to promote differential association of the protein to either the 70S or 30S ribosomal species. Exactly how guanine nucleotide binding prompts a change in the association of this protein with the ribosome is not understood. The switch regions of most GTPases are highly flexible and cannot be observed by crystallography. Therefore, molecular dynamics simulations were used to visualize these regions of BipA in the various guanine nucleotide bound states. The ppGpp simulation suggests that a conserved arginine, located in the switch I region of the protein, makes direct contact with the alarmone. To examine whether this residue could be a specificity determinant for ppGpp binding, steady state kinetics and isothermal titration calorimetry measurements were done to assess how an alanine substitution at this site would impact the biochemical properties of BipA. These experiments revealed that although the GTP hydrolysis and GTP binding properties of the protein were unaltered, ppGpp binding was greatly diminished. In contrast to the wild-type protein, filter binding assays show that the alanine-substituted BipA does not associate with 30S ribosomes in the presence of ppGpp. Hydrogen deuterium exchange mass spectrometry confirm that the introduction of this mutation changes the overall dynamics of the protein away from the BipA:ppGpp state and thus selectively abolishing association with the ribosome. In bacteria, this mutant is unable to recover from the onset of stress, confirming the requirement of ppGpp binding by BipA for bacteria to mount an adaptive response via the formation of the 30S:BipA complex.