EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer

Abstract

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know 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 ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

Keywords: ABC Transporter; Electron Paramagnetic Resonance (EPR); Importer; Membrane Proteins; Molybdate; Protein Conformation; Protein Dynamics.

FIGURE 1.

CW-EPR spectroscopy of TM5a residues showing nucleotide-dependent mobility changes at the periplasmic gate. A, MolB residues S180C, L185C, and D173C (shown as red sticks) were spin-labeled with MTSL. TM5 and TM5a are colored cyan or purple according to the TMD monomer. B, CW-EPR spectra for S180C + MTSL (100-G scan width), L185C + MTSL (150 G), and D173C + MTSL (100G). Blue lines, apo state; red lines, ATP-bound state. Mobile and immobile components are identified for the S180C spectra. Peak broadening (induced by close proximity between spin label pairs) is identified for L185C; note that the amount of broadening is the same for all L185C spectra. C, post-hydrolysis spectra (green) of both mutants overlaid with the respective apo spectra. Overlaid spectra have been normalized for equal spin (normalized double integration values).

FIGURE 2.

CW-EPR spectroscopy showing nucleotide-dependent mobility changes at the periplasmic gate in the presence of MolA. A, effect of MolA binding to MolBC mutants (purple) overlaid with apo spectra (blue). B, effect of ATP binding to the MolBC-A complex (cyan) overlaid with the apo-MolBC-A spectra. C, post-hydrolysis spectra (orange) of both mutant MolBC-A complexes. Overlaid spectra have been normalized for equal spin (normalized double integration values).

FIGURE 3.

Effect of ligand on disulfide cross-linking at the periplasmic gate and cytoplasmic gate I. A, two residues at the periplasmic gate (Leu-178 and Val-182) and one residue at the cytoplasmic side of TM5 (Leu-148) are shown as red sticks. Residues mutated to cysteines formed a disulfide cross-link across the translocation pathway. B, three different conditions (apo, MolA-bound, and BeF_x/ATP-trapped) were tested for cross-linking at the three sites relative to a Cys-less control. Cross-linking was observed by monitoring the gel shift from monomeric TMD (30 kDa) to dimeric TMD (60 kDa). The bottom band at 24 kDa is the NBD, and the PBP (MolA) is 39 kDa. C, cross-linking was quantified by image analysis (ImageJ software) of the SDS-polyacrylamide gel in B. In-gel dimer formation of Cys-less MolB was likewise quantified and used to correct the cross-linking values of the mutants. For better comparison with cross-linking percentages, the specific activity of each mutant was calculated as a percent of Cys-less specific activity and subtracted from 100 to give the percent inhibition. Error bars were calculated from the standard error of specific activity measurements (n = 3).

FIGURE 4.

CW-EPR spectroscopy of cytoplasmic gate I showing ATP-dependent conformational change. A, I151C of MolB shown as red sticks. B, CW-EPR spectra for I151C + MTSL were collected over a wide scan range (250 G) due to extensive broadening seen in the apo spectrum (blue). The overlaid ATP-bound spectrum (red) shows spin decoupling. Spectra have been normalized for equal spin (normalized double integration values). C, ATP hydrolysis (green) returns spin labels at I151C to an apo-like state. D–F, CW-EPR spectra of MolBC and MolBC-A overlaid in the apo, ATP-bound, and post-hydrolysis states, respectively.

FIGURE 5.

Nucleotide-dependent movement of cytoplasmic gate II shown by CW-EPR spectroscopy and disulfide cross-linking. A, three residues along the TM2-TM3 loop (Asn-89, Leu-91, and Asn-93, shown as red sticks) were separately mutated to cysteines. B, CW-EPR spectra for N93C + MTSL were collected over 250 G. All spectra were normalized for equal spin (normalized double integration values). The apo (blue) and ATP-bound (red) spectra are overlaid to highlight the broadening of the ATP-bound spectrum, indicative of spin coupling. C, the line shape returns to an apo-like state upon the addition of MgCl₂ (green). D, four conditions (apo, MolA-bound, BeF_x/ATP-trapped, and MolA/BeF_x/ADP-Mg²⁺-trapped) were tested for TMD cross-linking at each site. Cross-linking may be quantified by monitoring the gel shift from monomeric TMD (30 kDa) to dimeric TMD (60 kDa). A Cys-less control was prepared in parallel under each condition and run on a gel to check for dimer formation of MolB.

FIGURE 6.

Proposed mechanism of MolBC-A. A, the first state represents the inward-facing conformation seen in the MolBC crystal structure. B, clipped top-down view of MolB showing the two cytoplasmic gates. After binding loaded MolA and ATP, the periplasmic gate will unlock (making it more permeable), cytoplasmic gate I will open farther, and gate II will close. C, substrate may slip through the unlocked periplasmic gate into a putative translocation chamber. When ATP is hydrolyzed, the periplasmic gate locks, preventing backflow, and cytoplasmic gate II reverts to an open conformation, allowing substrate to enter the cytoplasm. For functional transport, unloaded MolA must depart MolBC, returning the transport cycle to the initial state.