Biology subjects

Ford, R. C.

Publications and source records attributed to Ford, R. C..

4 recordsLinked to original sources

bioRxiv2025

Insights into the linker domain in ABCB1/P-glycoprotein

ABCB1/P-glycoprotein(P-gp) is one of the best-known ABC transporters and its involvement in the clearance of chemotherapy agents in cancer cells can lead to multidrug resistance. The N- and C-terminal halves of P-gp are joined by a flexible linker region. Linker regions in ABC transporters often play pivotal roles (i.e. CFTR), however, there is no consensus on the importance of the role of the linker, the complete 3D-structure of which has not yet been determined. Prior studies on linker length showed major effects on P-gp function. In this work, we combined microsecond simulations ({approx}50{micro}s) on mouse and human P-glycoprotein, cryo-EM and other computational methods to trace a profile of the linker region. Different experimentally verified conditions involving the linker region were simulated, analysed and compared. Attachment of the linker to the membrane was spontaneously and occasionally encountered via a recurrent series of amino-acids, despite random starting conformations, suggesting that the lipid bilayer might be involved in linker regulation. As carried out in previous experiments, shortening the linker could potentially disconnect the ATPase activity from the substrate transport by interfering with one ATP site while introducing a short partially-structured linker shows remarkable stability in accelerated MD. Finally, we investigated the linker region by positioning it in specific quadrants of the 3D space, with simulations suggesting 1) a possible influence on protein flexibility, 2) selective tightening and opening of the two halves of P-gp, and a 3) possible stabilization of the inward-facing/occluded-state. The interaction between the linker and ATPs was investigated using protein-protein docking, simulations and a re-refined cryo-EM map where medium-low resolution and weak Coulomb density was found between the nucleotide binding domains.

biochemistry↗

bioRxiv2021

Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor.

ABCB1 (P-glycoprotein) is an ATP binding cassette transporter that is involved in the clearance of xenobiotics and it affects the disposition of many drugs in the body. Here we have studied ABCB1 in the drug-bound and drug-free states, simultaneously, using high contrast cryo-electron microscopy imaging and a Volta phase plate. The binding of the potent CFTR potentiator, ivacaftor, at a site in the central aqueous cavity is mediated by transmembrane -helices 3,6,10,11 & 12. Binding is associated with a wider separation of the two halves of the transporter in the inward-facing state. Induced-fit changes the nucleotide binding domains in a way that may explain their increased affinity for ATP when drug is bound. Comparison of ivacaftor-bound structures of CFTR and ABCB1 suggests common features in the binding modes.

biochemistry↗

bioRxiv2019

In-vivo crystals reveal protein interactions

Crystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at -1 and -3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

biochemistry↗

bioRxiv2018

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9A resolution

P-glycoprotein (ABCB1) is a ATP-binding cassette transporter that plays an important role in the removal of drugs and xenobiotic compounds from the cell. It is also associated with multi-drug resistance in cancer. Here we report novel features of the cryo-EM-derived structure of P-glycoprotein in the post-hydrolytic state: The cytosolic nucleotide-binding domains (NBDs) are separated despite ADP remaining bound to the NBDs. Gaps in the TMDs that connect to the inner hydrophilic cavity are back-filled by detergent head-groups from the annular detergent micelle and are close to two regions predicted to delineate two pseudo-symmetry-related drug-binding sites. In this conformation, the (newly-resolved) N-terminal extension, NBD-TMD linker region and gap-filling detergents all appear to impede NBD dimerisation. We propose a model for the mechanism of action of the exporter where ATP will be bound to the protein for most of the time, consistent with the high physiological ATP concentrations in vivo.

biochemistry↗