Coupling of Calcium and Substrate Binding through Loop Alignment in the Outer-Membrane Transporter BtuB

Abstract

In Gram-negative bacteria, TonB-dependent outer-membrane transporters bind large, scarce organometallic substrates with high affinity preceding active transport. The cobalamin transporter BtuB requires the additional binding of two Ca 2+ ions before substrate binding can occur, but the underlying molecular mechanism is unknown. Using the crystallographic structures available for different bound states of BtuB, we have carried out extended molecular dynamics simulations of multiple functional states of BtuB to address the role of Ca 2+ in substrate recruitment. We find that Ca 2+ binding both stabilizes and repositions key extracellular loops of BtuB, optimizing interactions with the substrate. Interestingly, replacement by Mg 2+ abolishes this effect, in accordance with experiments. Using a set of new force-field parameters developed for cyanocobalamin, we also simulated the substrate-bound form of BtuB, where we observed interactions not seen in the crystal structure between the substrate and loops previously found to be important for binding and transport. Based on our results, we suggest that the large size of cobalamin compared to other TonB-dependent transporter substrates explains the requirement of Ca 2+ binding for high-affinity substrate recruitment in BtuB.