In Escherichia coli, vitamin B12 is transported via the TonB dependent outer membrane transporter BtuB. Structural models show that this protein contains an N-terminal core domain folded inside a β barrel; however, the exact mechanism of vitamin B12 transport is unclear. Ion pairs frequently mediate membrane protein function and there are many ion pairs between the core and barrel domains of BtuB. These potentially act as conformational switches that might function during vitamin B12 transport and/or during a transmembrane signaling process. When substrate binds BtuB, a highly conserved region of the core, called the Ton box, unfolds and expands into the periplasm. Previously, our lab examined an ion pair near the Ton box (R14- D316) using two mutant cycle analysis coupled with side-directed spin labeling and electron paramagnetic resonance spectroscopy (EPR) . Mutating residues in the ion pair to alanine unfold the Ton box even without the substrate; demonstrating this interaction is critical to this substrate-driven signaling process. Currently, we are investigating the role of five conserved ion pairs between the two domains (R69-E419, R111-E465, D53-R526, R36-D515, R47-D548). In contrast to previous results, all the studied ion pair mutants are found to inhibit Ton box unfolding in the presence of the substrate. However, the extent of the inhibition is different between these mutants. In the future, we will extend these measurements to determine the network of residues that mediate signaling and transport in BtuB.

Conserved ion pairs between the barrel and hatch domain of BtuB are required for vitamin B12 transport and/or during transmembrane signaling

254th National Meeting (2017)

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