RESUMO
The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147-239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB(147-239) was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B12) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C8E4 (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2(1)2(1)2(1) (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 angstroms). The asymmetric unit consists of a single BtuB-TonB complex. Data sets have been collected to 2.1 angstroms resolution at a synchrotron beamline (APS SER-CAT 22-ID).
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Sítios de Ligação , Cálcio/metabolismo , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Vitamina B 12/metabolismoRESUMO
In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a beta strand that is recruited to the existing beta sheet of TonB, which is consistent with a mechanical pulling model of transport.
