College of Liberal Arts & Sciences

Pulling, pushing and cross-linking towards a mechanism of active transport across the Gram negative outer membrane

Tuesday, May 3, 2016

Tuesday, May 3, 2016
12:30 pm, MRB 200 Conference Room

Dr. Robert Nakamoto

Department of Molecular Physiology & Biological Physics and the Center for Membrane Biology, University of Virginia

Pulling, pushing and cross-linking towards a mechanism of active transport across the Gram negative outer membrane

The Gram negative bacteria TonB-dependent transporters (TBDT) are responsible for the initial binding and import of a variety of metal complexes across the outer membrane. An amino-terminal lumenal domain fits within the 22-stranded β-barrel and contains the determinants for substrate binding. Current models suggest that the TBDT carries out active transport driven by energy from the proton motive force (pmf), which is transmitted via direct interactions between the inner membrane protein TonB and a TBDT N-terminal motif called the Ton Box. The lumenal domain must undergo large conformational changes to accommodate passage of the substrate through the barrel, but the molecular mechanism is unknown. We show that disulfide bonds can be induced to form between pairs of cysteines introduced at barrel-lumenal domain contact points, consistent with the x-ray crystallographic structures. However, we also find spontaneously formed, stoichiometric cross-links between other pairs of cysteines indicate a lumenal domain conformation distinct from the x-ray structures. Three different TBDTs with cysteines in equivalent positions all form spontaneous disulfides. The unexpected cross-links block transport and activity is recovered upon reduction. In at least one cross-linked example, the E. coli BtuB binds its substrate vitamin B12 in outer membrane fragments but not in whole cells, indicating that the substrate binding site of the cross-linked form is oriented towards the periplasmic side. In contrast, the transporter is oriented outwards in all other conditions, including in the absence of a pmf. These results suggest an alternating access transport mechanism.

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