Exploring the Solution Behavior of the Translational GTPase BipA Using Molecular Dynamics

BipA is a conserved prokaryotic GTPase necessary for securing bacterial survival and successful invasion of the host. Structural and biochemical studies indicate that GTP and ppGpp compete for binding to BipA to promote differential association of BipA to either the 70S or 30S ribosomal species. Exactly how guanine nucleotide binding to BipA prompts a change in the association of this protein with the ribosome is not understood. Crystallographic models show local structural rearrangements occur near the nucleotide-binding pocket but unexpectedly the overall domain arrangement, and therefore intramolecular contacts are similar in the various guanine nucleotide bound states. One explanation is that the lattice contacts in the crystal restrict the conformational space available to the protein. Another is that the BipA is metastable and binding of GTP or ppGpp trap intermediate states poised to bind the ribosome. This model would account for a reverse flow of information evidenced by the increase in BipA’s rate of GTP hydrolysis upon ribosome binding. We have monitored the solution dynamics of the various nucleotide bound states of BipA using amide hydrogen/deuterium exchange mass spectrometry (HDXMS). These data indicate that GTP and ppGpp binding lead to large scale conformational changes that are propagated throughout BipA, underscoring the idea that BipA is a metastable molecule where mutually exclusive association of GTP or ppGpp drive equilibria to alternate distinct conformations resulting in differential binding to the ribosome. Molecular dynamics simulations support this observation and point to dynamic allostery between the GTPase and novel C-terminal domain.