Biology subjects

O'Mara, M. L.

Publications and source records attributed to O'Mara, M. L..

6 recordsLinked to original sources

bioRxiv2026

Structural basis for inefficient G protein coupling by Frizzled receptors

Frizzleds (FZDs) are class F G protein-coupled receptors (GPCRs) that primarily signal through Dishevelled (DVL) to initiate Wnt signalling, with evidence for context-specific coupling to heterotrimeric G proteins. Every FZD-G protein structure to date has been determined without an agonist, implying that agonist binding is not required for stable complex formation in vitro and directly contradicting the difficulty of demonstrating G protein coupling in cells. All these structures contain unassigned density between the receptor and the heterotrimeric G protein. From structures generated by single-particle cryo-electron microscopy (cryo-EM) of FZD7-Gs and FZD5-Gq, we identify this density as the detergent lauryl maltose neopentyl glycol (LMNG), which bridges the receptor and G protein to stabilise a weak, shallow interface. Absence of LMNG - in biochemical preparations and molecular dynamics simulations - destabilises the complex, showing that constitutive FZD-G protein engagement is intrinsically weak. The weak G protein coupling is governed by a conserved intracellular interface that when mutated differentially influences DVL and G protein signalling. Our findings show that LMNG has masked the instability of purified FZD-G protein complexes and provide the structural basis of FZDs as inherently weak G protein activators that are predisposed to DVL signalling.

biochemistry↗

bioRxiv2026

Structural basis of drug efflux by the staphylococcal efflux pump QacA

The QacA DHA2 exporter from Staphylococcus aureus is a prototypical multidrug transporter that, like other bacterial efflux pumps, can extrude a wide range of cytotoxic compounds thus playing a crucial role in antimicrobial resistance. Here, we report crystal structures of wild-type QacA in three key conformational states: inward-open, outward-open and ethidium-bound, representing the first ligand-bound structure of a 14 transmembrane helices (TM) DHA2 transporter. In combination with computational and functional studies, these structures provide a mechanistic framework to understand drug recognition and extrusion. Structural analyses reveal remarkable adaptability within the binding pocket, including a ligand-induced deformation of TM5 that enables coordination of ethidium bromide in the outward-open state. Molecular dynamics simulations show spontaneous lipid entry into the transporter core and suggest that substrate binding from the inner membrane leaflet initiates a conformational transition to an outward-open state, stabilizing high-affinity interactions. Subsequent binding site protonation drives substrate extrusion. Together, these findings elucidate the structural dynamics and mechanistic underpinnings of QacA-mediated multidrug transport, highlighting conformational flexibility and proton-coupled electrostatic changes as key determinants of multidrug recognition and extrusion. This study provides a foundational framework for developing targeted inhibitors to combat bacterial multidrug resistance.

biochemistry↗

bioRxiv2025

Structural insights into allosteric mechanism of glycine transporter-mediated analgesia

Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2-82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2-82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2-82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.

neuroscience↗

bioRxiv2022

Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2

Membrane cholesterol binds to and modulates the function of the specific SLC6 transporters. Here we investigate how cholesterol binds to and modulates the rate of glycine transport by the SLC6 glycine transporter GlyT2, and how this impacts lipid inhibition of GlyT2. Bioactive lipid inhibitors of GlyT2 are analgesics that bind to the lipid allosteric site of the outward facing GlyT2 conformation that is accessible from the extracellular solution. Using molecular dynamics simulations, mutagenesis and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a cholesterol binding site formed by transmembrane helices 1, 5 and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3 hydroxyl group into the cholesterol binding cavity to interact with the base of the lipid allosteric site and the bound inhibitor. The recruitment of membrane cholesterol by allosteric GlyT2 inhibitors is a potential avenue for the development of high-potency, specific pain analgesics and could provide alternative therapeutics that target GlyT2 and other SLC6 neurotransmitter transporters.

neuroscience↗

bioRxiv2021

Lipid fingerprints are similar between SLC6 transporters in the neuronal membrane

We use molecular dynamics simulations to characterise the local lipid annulus, or "fingerprint", of three SLC6 transporters (dDAT, hSERT, and GlyT2) embedded into a complex neuronal membrane. New membrane analysis tools were created to improve leaflet detection and leaflet-dependent properties. Overall, lipid fingerprints are comprised of similar lipids when grouped by headgroup or tail saturation. The enrichment and depletion of specific lipids, including sites of cholesterol contacts, varies between transporters. The subtle differences in lipid fingerprints results in varying membrane biophysical properties near the transporter. Through comparisons to previous literature, we highlight that the lipid-fingerprint in complex membranes is highly dependent on membrane composition. Furthermore, through embedding these transporters in a simplified model membrane, we show that the simplified membrane is not able to capture the biophysical properties of the complex membrane. Our results further characterise how the presence and identity of membrane proteins affects the complex interplay of lipid-protein interactions, including the local lipid environment and membrane biophysical properties. HIGHLIGHTSO_LILipid fingerprints are comprised of similar lipid classes C_LIO_LISites of specific lipid contacts, including CHOL, varies between transporters C_LIO_LIChanges in lipid annulus result in variable local membrane biophysical properties C_LIO_LIMembrane composition, including that of complex membranes, affects lipid annulus C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/427530v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@ae6ee9org.highwire.dtl.DTLVardef@1f39af0org.highwire.dtl.DTLVardef@412256org.highwire.dtl.DTLVardef@355f1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

The inhibitory activity of GlyT2 targeting bioactive lipid analgesics are influenced by formation of a deep lipid cavity

The human glycine transporter 2 (GlyT2 or SLC6A5) has emerged as a promising drug target for the development of new analgesics to manage chronic pain. N-acyl amino acids inhibit GlyT2 through binding to an allosteric binding site to produce analgesia in vivo with minimal overt side effects. In this paper we use a combination of medicinal chemistry, electrophysiology, and computational modelling to explore the molecular basis of GlyT2 inhibition at the allosteric site. We show how N-acyl amino acid head group stereochemistry, tail length and double bond position promote enhanced inhibition by deep penetration into the binding pocket. This work provides new insights into the interaction of lipids with transport proteins and will aid in future rational design of novel GlyT2 inhibitors.

biochemistry↗