Biology subjects

Bushell, S. R.

Publications and source records attributed to Bushell, S. R..

4 recordsLinked to original sources

bioRxiv2023

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes which link substrate release and opening of the cytoplasmic gate, and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

biophysics↗

bioRxiv2020

The structural basis of fatty acid elongation by the ELOVL elongases

Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinsons, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 [A] long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

biochemistry↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗

bioRxiv2018

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic lipid-altered tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.\n\nHighlightsO_LIStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis\nC_LIO_LIDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity\nC_LIO_LIStructures, kinetics and biosynthesis reveal role of lipid in tunicamycin\nC_LIO_LILipid-altered, tunicamycin analogues give non-toxic antibiotics against TB\nC_LI

biochemistry↗