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

The periplasmic binding protein (PBP) BtuF plays a key role in transporting vitamin B-12 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 B-12 and BtuF recognition is still under debate. In the present work, conformational changes of BtuF upon B-12 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 B-12 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 B-12 proceed in a manner similar to the Venus flytrap mechanism. Our study therefore delineates a clear view of BtuF delivering B-12, 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.

Automated summary

The study investigated conformational changes of BtuF upon B-12 binding and release using hybrid approaches, highlighting the increased stability of BtuF with B-12 binding and the flexible regions within BtuF. The findings provide insights into the mechanism of B-12 recognition and deliverance by BtuF, showcasing the potential of hybrid methods in probing protein conformational dynamics.