E0029

Structure of the E. coli Outer Membrane Cobalamin Transporter BtuB. Michael Wiener, Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia, P.O. Box 800736, Charlottesville, VA 22908-0736.

All organisms require organometallic cofactors such as hemes, porphyrins and cobalamins, and must obtain them (or their precursors) from external sources. Gram-negative bacteria possess specialized systems that bind and transport these essential molecules across two membranes (outer and inner) and the periplasmic space for subsequent utilization within the cytosol. Specific outer membrane transporters function to bind their respective substrates in an energy-independent fashion. Following the binding event, energy-dependent transport across the outer membrane occurs. The outer membrane does not maintain an electrochemical gradient. The energy required for transport is obtained by coupling the outer membrane transporter to the inner membrane in a manner enabling utilization of the inner membrane’s protonmotive force. Many of these outer membrane transporters couple with the TonB protein, part of an inner membrane complex that transduces energy to drive active transport. In these TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved four crystal structures of BtuB: an initial methionine-substitution mutant used for experimental phase determination, the wildtype protein, wildtype protein with bound calcium ions, and wildtype protein with bound calcium and cyanocobalamin (vitamin B₁₂) substrate. In these structures, the Ton box is ordered and undergoes conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K_d ~ 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.