Coupling of calcium and substrate binding through loop alignment in the outer-membrane transporter BtuB

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• TonB-dependent transporters in Gram-negative bacteria mediate high-affinity binding of scarce organometallic substrates.
• The cobalamin transporter BtuB requires Ca(2+) ions for substrate binding, but the mechanism remains unclear.

Purpose of the Study:

• To elucidate the molecular mechanism by which Ca(2+) facilitates substrate recruitment by the BtuB transporter.
• To investigate the role of Ca(2+) in stabilizing BtuB for optimal substrate interaction.

Main Methods:

• Extended molecular dynamics simulations of multiple functional states of BtuB.
• Utilized crystallographic structures of BtuB in different bound states.
• Developed new force-field parameters for cyanocobalamin to simulate substrate-bound BtuB.

Main Results:

• Ca(2+) binding stabilizes and repositions key extracellular loops of BtuB, enhancing substrate interaction.
• Mg(2+) cannot replace Ca(2+) in mediating these stabilizing effects.
• Simulations revealed unobserved substrate-loop interactions crucial for binding and transport.

Conclusions:

• Ca(2+) binding is essential for high-affinity substrate recruitment by BtuB.
• The large size of cobalamin likely necessitates Ca(2+) for efficient binding compared to other TonB-dependent transporter substrates.