Molecular Basis of Transmembrane Signaling in a Membrane Transporter

Abstract

Since there is no source of energy at the outer membrane, the active uptake of essential nutrients by outer membrane transporters in gram-negative bacteria is dependent on coupling to the electrochemical gradient across the cytoplasmic membrane via the protein TonB. The E.coli outer membrane vitamin B12 transporter BtuB and its paralogs undergo a substrate-dependent conformational transition in a conserved periplasmic motif called the Ton box, which is the recognition element for TonB and presumably a transmembrane signal that the transporter is ready to receive the energy required for substrate transport. We have identified several key residues that are important in this transmembrane signaling mechanism using a combination of mutagenesis, site-directed spin labeling EPR, and X-ray crystallography. Furthermore, we examined the effect of transport-defective mutations on the structure of the Ton box and substrate-binding site, and using and EPR-based binding assay we also determined the effect of these mutations on the BtuB-TonB association. Collectively, the results have been evaluated in terms of a model for transmembrane signaling in BtuB.