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 E. coli 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.