Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry.

Nicotinamide nucleotide transhydrogenases are integral membrane proteins that utilizes the proton motive force to reduce NADP to NADPH while converting NADH to NAD. Atomic structures of various transhydrogenases in different ligand-bound states have become available, and it is clear that the molecular mechanism involves major conformational changes. Here we utilized hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map ligand binding sites and analyzed the structural dynamics of transhydrogenase. We found different allosteric effects on the protein depending on the bound ligand (NAD, NADH, NADP, NADPH). The binding of either NADP or NADPH to domain III had pronounced effects on the transmembrane helices comprising the proton-conducting channel in domain II. We also made use of cyclic ion mobility separation mass spectrometry (cyclic IMS-MS) to maximize coverage and sensitivity in the transmembrane domain, showing for the first time that this technique can be used for HDX-MS studies. Using cyclic IMS-MS, we increased sequence coverage from 68 % to 73 % in the transmembrane segments. Taken together, our results provide important new insights into the transhydrogenase reaction cycle and demonstrate the benefit of this new technique for HDX-MS to study ligand binding and conformational dynamics in membrane proteins.