To understand how the gates of the transmembrane domains are repositioned to allow small substrates into the cell, we used electron paramagnetic resonance spectroscopy (EPR) to capture a snapshot of the MolB2C2-A complex at each point in the transport mechanism. We identified four states, from apo to post-ATP hydrolysis, that the transporter must undergo to translocate substrate (Figure A) .
In the absence of nucleotide, the periplasmic gate is locked (closed
) and the cytoplasmic gates are open (State 1). When ATP binds, the NBDs close around the nucleotide. This classical conformational change of the NBDs rearranges the TMD helices of MolBC, which allows the periplasmic gate to unlock and permits substrate to slip into the translocation pathway (State 2). Concurrently, when ATP is bound, the cytoplasmic gate II closes and gate I is forced to a widely separated conformation. We speculate that a translocation chamber is formed when cytoplasmic gate II is closed and binding protein caps the unlocked periplasmic gate (State 3). After ATP hydrolysis, the periplasmic gate locks and the cytoplasmic gates assume an apo-like, partially open state (State 4).
These results are significant because they reveal that the mechanism of a Type II transporter for a small substrate (molybdate) differs from the mechanism of the Type II transporters of large substrates (vitamin B12 BtuCD). Unlike BtuCD (Figure B), the translocation pathway of MolBC opens just enough to allow through a small substrate like molybdate, suggesting that in addition to the SBP, the size of the translocation pathway size provides an additional selectivity filter; thus, the transport mechanisms of ABC importers are more mechanistically diverse than previously thought.