Structure, Function, and Application of Bacterial ABC Transporters

Citation

Fan, Chengcheng (2020) Structure, Function, and Application of Bacterial ABC Transporters. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5t65-0047. https://resolver.caltech.edu/CaltechTHESIS:06012020-160106726

Abstract

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single particle cryo-EM in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione binding site and the associated substrate binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO₄ eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provides an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide binding domains in the inward-facing conformation.

The ATPase kinetic data was fit to a non-essential activator model with expansion to two substrate binding sites. While the structural data suggests that MgATP and GSSG bind to distinct states, outward- and inward-facing conformations, respectively, and hence might be expected to exhibit negative cooperativity, the kinetic data support a more complex interplay and also the importance of lipid molecule presence. How GSSG binding stimulates ATPase activity remains an open question and highlights the importance of the still elusive ternary complex with both MgATP and GSSG bound to NaAtm1.

Besides the structural and functional characterizations of the ABC exporter, NaAtm1, we additionally determined crystal structures of the repurposed periplasmic binding protein (PBP) from the ABC importer system. These PBPs are designed with circularly permutated GFP to act as biosensors to sense the concentrations of smoking cessation drugs and neurotransmitters under cellular conditions. The crystal structures determined for the nicotine and acetylcholine biosensors not only revealed the key residues in ligand binding, but also demonstrated similar ligand induced conformational changes as seen in other PBPs by following the Venus-flytrap mechanism.

Thesis Files