Coordinated Rearrangements between Cytoplasmic and Periplasmic Domains of the Membrane Protein Complex ExbB-ExbD of Escherichia coli

• Membrane proteins ExbB and ExbD form a complex of 4:2 stoichiometry
• The protein-detergent complex is ∼240 kDa: ∼140 kDa protein + ∼100 kDa DDM
• 2D EM analyses reveal structural diversity despite biochemical homogeneity
• 3D reconstructions show different structural states and coordinated rearrangement

SummaryGram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions.

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