Biology subjects

Brotherton, D. H.

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

2 recordsLinked to original sources

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↗