Biology subjects

Colyer, A.

Publications and source records attributed to Colyer, A..

4 recordsLinked to original sources

bioRxiv2026

PqiABC forms a membrane-bridging conduit and mediates bidirectional phospholipid transport across the Gram-negative bacterial envelope

The mechanism by which glycerophospholipids are transported between the inner and outer membranes in Gram-negative bacteria remains poorly understood. In Escherichia coli, the paraquat-inducible (Pqi) pathway, comprising the inner membrane protein PqiA, the periplasm-spanning MCE-family protein PqiB, and the outer membrane lipoprotein PqiC, has been implicated in this process. These components are proposed to assemble into a quaternary complex that forms a continuous channel bridging the inner and outer membranes. Here, using neutron reflectometry and quartz crystal microbalance with dissipation monitoring, we perform a dynamic structural analysis of PqiABC within a planar double bilayer membrane-mimetic system. This approach reveals that PqiABC assembles into a stable, envelope-spanning complex anchored to both membranes, consistent with its proposed conduit architecture. Furthermore, using neutron reflectometry in combination with complementary fluorescence-based assays, we demonstrate that PqiABC mediates passive glycerophospholipid transport, supporting bidirectional lipid exchange between membranes. Together, these findings establish PqiABC as a membrane-bridging lipid transport system and provide direct evidence for a mechanism of passive glycerophospholipid equilibration across the bacterial envelope.

biophysics↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2025

Allostery links hACE2 binding, pan-variant neutralisation and helical extension in the SARS-CoV-2 Spike protein

The SARS-CoV-2 spike protein is highly antigenic, with epitopes in three distinct regions of the receptor binding domain (RBD) alone that have known mechanisms of neutralization. In previous work, we predicted a fourth RBD epitope based on allosteric conformational perturbations measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS) upon complexation with the canonical spike protein target, human angiotensin-converting enzyme 2 (hACE2). We subsequently identified a pan-neutralizing antibody (ICO-hu104) with the predicted epitope, however, as the epitope was somewhat distant from the hACE2 binding interface, and our previous work limited to the spike RBD, the neutralization mechanism was unclear. Using HDX-MS, we investigated the binding of ICO-hu104 to the full-length SARS-CoV-2 spike protein from Wuhan, Delta and Omicron variants. We demonstrate that binding of ICO-hu104 at its epitope results in an increase in deuterium uptake in the distant HR1 domain for variants of concern, which in a biological context could be indicative of destabilisation of the helices within this region, promoting S1 shedding or failure of helical extension during S2-mediated fusion. This is supported by our computational modelling, highlighting propagation of allosteric effects to the S2 coiled-coil region. Collectively, this work demonstrates an alternative neutralization mechanism for ICO-hu104 which is distinct from its first-generation predecessors and thus opens alternative avenues targeting non-RBD epitopes through assessment of allosteric perturbations. HighlightsO_LIHDX-MS reveals decreased deuterium uptake within the HR1 region of S2 for SARS-CoV-2 spike protein for variants of concern when bound to ICO-hu104. C_LIO_LIComputational modelling validates high allosteric coupling between ICO-hu104 epitope and HR1 region. C_LIO_LISuggests an alternative neutralization mechanism from its predecessor ICO-hu23, whereby destabilisation of helices within the HR1 region promotes S1 shedding and/or failure of helical extension during S2-mediated fusion. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/635280v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1175bdborg.highwire.dtl.DTLVardef@8fb17borg.highwire.dtl.DTLVardef@1cd13f7org.highwire.dtl.DTLVardef@d9c121_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗