Conformational changes and binding property of the periplasmic binding protein BtuF during vitamin B12 transport revealed by collision-induced unfolding, hydrogen-deuterium exchange mass spectrometry and molecular dynamic simulation

Abstract

The periplasmic binding protein (PBP) BtuF plays a key role in transporting vitamin B₁₂ from periplasm to the ATP-binding cassette (ABC) transporter BtuCD. Conformational changes of BtuF during transport can hardly be captured by traditional biophysical methods and the exact mechanism regarding B₁₂ and BtuF recognition is still under debate. In the present work, conformational changes of BtuF upon B₁₂ binding and release were investigated using hybrid approaches including collision-induced unfolding (CIU), hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulation. It was found that B₁₂ binding increased the stability of BtuF. In addition, fast exchange regions of BtuF were localized. Most importantly, midpoint of hinge helix in BtuF was found highly flexible, and binding of B₁₂ proceed in a manner similar to the Venus flytrap mechanism. Our study therefore delineates a clear view of BtuF delivering B₁₂, and demonstrated a hybrid approach encompassing MS and computer based methods that holds great potential to the probing of conformational dynamics of proteins in action.

Keywords: Collision induced unfolding; Conformational change; Hydrogen-deuterium exchange; Mass spectrometry; Molecular dynamics simulation; Periplasmic binding protein.