Biology subjects

Brotherton, D. H.

Publications and source records attributed to Brotherton, D. H..

6 recordsLinked to original sources

bioRxiv2026

Structural insight into sodium-dependent bile acid transport by members of the SLC10 family

ASBT and NTCP are sodium-coupled secondary transporters of the SLC10 family that play critical roles in the enterohepatic recycling of bile acids. Secondary transporters generally function through the alternating access mechanism. However, in outward-facing structures of NTCP this mechanism is violated with a pore running through the protein. No such pore is observed in previously reported structures of bacterial ASBT homologues, but these proteins have an additional transmembrane-helix adjacent to the binding site. Here we investigate another homologue of ASBT without this extra helix. We solve the crystal structure of the inward-facing state in multiple conformations and a humanised version in the outward-facing state, with and without bile acid bound. Unlike for NTCP there is no pore through the protein as the exit is blocked by the flexible TM6. Consequently, the presence of the pore is not necessarily common to the family. Molecular dynamics simulations of both bacterial and human proteins highlight that while the bile acid substrate is anchored to residues at the centre of the transporter, lipids from the surrounding membrane interact with the hydrophobic sterol group. Incorporating lipids into the transport mechanism can explain how bile acids modified by large hydrophobic groups can bind and be transported.

biochemistry↗

bioRxiv2026

Structure of Connexin26 from Lepidosiren paradoxa

Connexin gap junction channels enable the direct exchange of molecules and ions between cells. Channel opening is regulated by various physiological stimuli. Human connexin26 gap junction channels close in response to elevated levels of CO2 in a process that is independent of pH, with structures demonstrating a [CO2]-dependent conformational change. Cx26 from the lungfish, Lepidosiren paradoxa is also CO2 sensitive. Here we solve its structure at high [CO2]. We observe an open conformation of the protein where the N-terminal helix that influences the aperture of the pore is pulled away from the centre. This conformation is stabilised by the presence of a detergent binding between TM3 and TM4 that would prevent the conformational changes necessary to close the protein upon exchange to high [CO2]. The structure supports a mechanism in which the conformation of a motif shown to be important for CO2 sensitivity is correlated with the opening of the pore.

biochemistry↗

bioRxiv2023

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

biochemistry↗

bioRxiv2023

Mechanism of substrate binding and transport in BASS transporters

The Bile Acid Sodium Symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here we solve the crystal structure at 2.3 [A] of a transporter from Neisseria Meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM. The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

biochemistry↗

bioRxiv2021

Structure and mechanism of a cytosine transport protein

CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB also to be important in the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence in the organism. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4[A] resolution in complex with cytosine. We show that the protein carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 make specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.

biochemistry↗

bioRxiv2020

Conformational changes and channel gating induced by CO2 binding to Connexin26

CO2 is the inevitable by-product of oxidative metabolism. Many physiological processes such as breathing1 and cerebral blood flow2 are sensitive to CO2. Historically, the physiological actions of CO2 have been regarded as being mediated exclusively via changes in pH. Here, we change this consensus by showing that the gap junction protein Connexin26 (Cx26) acts as a receptor for CO2 showing sensitivity to modest changes in PCO2 around the physiological norm3-6. Mass spectrometry analysis7 shows that CO2 carbamylates specific lysines on a regulatory loop of Cx26 at high, but not at low levels of PCO2. By means of high resolution cryo-EM, we have solved structures of Cx26 gap junctions at 1.9, 2.2 and 2.1 [A] for PCO2 of 90, 55 and 20 mmHg respectively, all at pH 7.4. Classification of the particles at each level of PCO2, shows the transmembrane helices and N-terminal helix flexing at the dynamic cytoplasmic side of the protein. Gating of Cx26 gap junctions by CO2 involves movements of the N-terminus to plug the channel at high PCO2. We therefore provide mechanistic detail for a new paradigm by which CO2 can directly control breathing8 and other key physiological functions9.

biochemistry↗