EPR Spectroscopy of MOLB2C2-A Reveals Mechanism of Transport for A Type II Molybdate Importer

Abstract

ABC importers are embedded in the plasma membrane of all bacteria. There they facilitate the uptake of several vital nutrients and cofactors. Due to differences in structure and activity, ABC importers are divided into two types. Type I importers have been well studied, but most of what we know about Type II importers has been observed in studies of the vitamin B12 importer, BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is about 10x smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC where alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed electron paramagnetic resonance (EPR) spectroscopy with the transporter imbedded in detergent micelles and liposomes (spherical lipid bilayers). The use of both detergent and lipid have allowed us to identify conformational changes in MolBC and the impact of the lipid environment. We observed that nucleotide binding and hydrolysis shift the conformation of MolBC at both the cytoplasmic and periplasmic gates. Mechanistic differences between MolBC and other Type II transporters highlight potential effects of substrate size on transport. Combining our results, we propose a mechanism for MolBC-A embedded in a lipid bilayer, which gives new insight in the transport of small substrates.