Coupling of Ca2+ and Substrate Binding in the Outer Membrane Transporter BtuB

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In Gram-negative bacteria, TonB-dependent transporters (TBDTs) in the outer membrane bind large, scarce organometallic substrates with high affinity preceding active transport. Unique among TBDTs, the cobalamin (e.g. vitamin B12) transporter BtuB requires the additional binding of two Ca2+ ions to its extracellular loops before high affinity substrate binding can occur. Using the wealth of crystallographic data available for BtuB, we have carried out extensive (over 200 ns) equilibrium molecular dynamics simulations of multiple functional states of BtuB to address the role of Ca2+ in substrate recruitment. Simulation of the apo structure of BtuB with Ca2+ ions present in solution demonstrated the fast, spontaneous recruitment of Ca2+ by BtuB. Once bound, we find that Ca2+ both stabilizes and repositions key loops, in order to optimize their interactions with the substrate. Interestingly, replacement by Mg2+ abolishes this effect, in accordance with experiments, due to its inability to stabilize the Ca2+-binding loops. We have also performed the first simulation of the substrate-bound form of BtuB using CHARMM forcefield parameters we recently developed for cyanocobalamin. In this simulation, we observe interactions between the substrate and two loops not seen in the crystal structures; however, these two loops have been reported to be important for substrate binding and transport. Based on our results, we suggest that the large size of cobalamin compared to other TBDT substrates explains the requirement of Ca2+ binding for high affinity substrate recruitment in BtuB but not other TBDTs. Research supported by NIH Grant 2R01-GM079800-06A2.