Biology subjects

Forrest, L. R.

Publications and source records attributed to Forrest, L. R..

15 recordsLinked to original sources

bioRxiv2026

Structural modelling and functional analysis support lipid binding by the dimeric E. coli DedA protein YqjA

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

biochemistry↗

bioRxiv2026

Cornichon receptors couple membrane adaptation to cargo selection during ER export.

Selective export of membrane proteins from the endoplasmic reticulum (ER) is fundamental for eukaryotic cell biology, yet how trafficking receptors coordinate cargo recognition with membrane adaptation and COPII recruitment remains unknown. Cornichon homolog (CNIH) proteins comprise a conserved family of trafficking receptors that mediate ER export of ion channels, G protein-coupled receptors (GPCRs), ATP-binding cassette (ABC) and solute carrier (SLC) transporters. Here, we determine cryo-electron microscopy structures of the prototypical cornichon receptor Erv14 bound to an SLC transporter in detergent and lipid nanodiscs. We show that cargo recognition is mediated by a dynamic network of interactions, in which structural lipids stabilize the receptor-cargo interface. Nanodisc structures reveal the assembly of a second Erv14 receptor that remodels the receptor-cargo interface in response to membrane architecture, thereby reducing local membrane thickness and providing direct structural evidence that cornichon receptors buffer hydrophobic mismatch during membrane protein biogenesis. Structural and trafficking analyses further show that the second receptor recruits the COPII adaptor Sec24, coupling membrane remodelling to cargo export. Together, our findings establish that cornichon receptors couple lipid-mediated membrane adaptation with cargo selection through sequential receptor assembly, linking membrane protein folding to selective COPII-mediated ER export. One sentence summaryCornichon receptors integrate membrane adaptation with cargo recognition to coordinate membrane protein quality control and selective ER export.

cell biology↗

bioRxiv2025

The Role of Chloride Ions in Serotonin Transport

The human serotonin (5-HT+) transporter SERT facilitates 5-HT+ transport into cells by coupling it to Na+ symport and K+ antiport. Although extracellular Cl- is also essential for transport, Cl-co-transport has been disputed, raising questions about the role of Cl- ions and why they are required. We show that Cl- gradients do not impact 5-HT+ accumulation, indicating that Cl- does not provide a driving force for uptake and arguing against stoichiometric Cl- symport. The presence of Cl- had only a small effect on Na+-mediated cytoplasmic pathway closure but markedly reduced the accessibility of residues in the extracellular pathway, consistent with modulation of the outward-facing states. Simulations illustrate that Cl- interacts strongly with a bound Na+ ion and stabilizes helix packing on the extracellular side. We propose that Cl- acts as an essential architectural cofactor by enhancing Na+ affinity and interactions between helices, thereby facilitating transport-related conformational transitions. Significance statementThe serotonin transporter (SERT) clears serotonin from synapses and is the target of widely used antidepressants. Although chloride ions are known to be required for SERT function, whether chloride is transported together with serotonin has remained unresolved for decades. We show that chloride gradients do not drive serotonin uptake, arguing against chloride symport. Instead, chloride stabilizes specific conformations of the transporter by strengthening sodium binding and extracellular helix packing. These findings redefine chloride not as a transported substrate, but as an architectural cofactor that facilitates conformational transitions required for transport. This reinterpretation reshapes our understanding of ion dependence in SERT and suggests a broader principle: ion requirements in related transporters may reflect structural stabilization rather than obligatory symport.

biochemistry↗

bioRxiv2023

Identification of the potassium binding site in serotonin transporter SERT

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter SERT, which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so- called Na2 site. Site directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT. Significance statementNeuronal signaling depends on efficient clearance of the neurotransmitter from the synaptic cleft. To this end, proteins such as serotonin transporter (SERT) leverage the gradients of Na+ and K+ ions across the cell membrane, generated by Na+/K+-ATPase. While the role of Na+ in neurotransmitter transport is well understood, our understanding of the role of potassium in SERT has been limited. In this study, the authors use a combination of biochemical, electrophysiological, and computational tools, to identify the Na2 site as the binding site for K+, shedding light on a critical aspect of neurotransmitter transport.

biochemistry↗

bioRxiv2023

Membrane free-energy landscapes derived from atomistic dynamics explain nonuniversal cholesterol-induced stiffening

All lipid membranes have inherent morphological preferences and resist deformation. Yet adaptations in membrane shape can and do occur at multiple length scales. While this plasticity is crucial for cellular physiology, the factors controlling the morphological energetics of lipid bilayers and the dominant mechanisms of membrane remodeling remain unclear. An ongoing debate regarding the universality of the stiffening effect of cholesterol underscores the challenges facing this field, both experimentally and theoretically, even for simple lipid mixtures. On the computational side, we have argued that enhanced- sampling all-atom molecular dynamics simulations are uniquely suited for quantification of membrane conformational energetics, not only because they minimize a-priori assumptions, but also because they permit analysis of bilayers in deformed states. To showcase this approach, we examine reported inconsistencies between alternative experimental measurements of bending moduli for cholesterol-enriched membranes. Specifically, we analyze lipid bilayers with different chain saturation, and compute free-energy landscapes for curvature deformations distributed over areas from [~]5 to [~]60 nm2. These enhanced simulations, totaling over 100 microseconds of sampling time, enable us to directly quantify both bending and tilt moduli, and to dissect the contributing factors and molecular mechanisms of curvature generation at each length scale. Our results show that cholesterol effects are lipid-specific, in agreement with giantvesicle measurements, and explain why experiments probing nanometer scale lipid dynamics diverge. In summary, we demonstrate that quantitative structure-mechanics relationships can now be established for heterogenous membranes, paving the way for addressing open fundamental questions in cell membrane mechanics. SignificanceElucidating the energetics and mechanisms of membrane remodeling is an essential step towards understanding cell physiology. This problem is challenging, however, because membrane bending involves both large-scale and atomic-level dynamics, which are difficult to measure simultaneously. A recent controversy regarding the stiffening effect of cholesterol, which is ubiquitous in animal cells, illustrates this challenge. We show how enhanced molecular-dynamics simulations can bridge this length-scale gap and reconcile seemingly incongruent observations. This approach facilitates a conceptual connection between lipid chemistry and membrane mechanics, thereby providing a solid basis for future research on remodeling phenomena, such as in membrane trafficking or viral infection.

biophysics↗

bioRxiv2022

Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used {micro}s-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport.

biochemistry↗

bioRxiv2022

Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation

The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.

biophysics↗

bioRxiv2020

Refining pairwise sequence alignments of membrane proteins by the incorporation of anchors

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly aligned by a standard pairwise alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the alignment. An alternative approach is the use of restraints, or anchors, to incorporate such position-matching explicitly during alignment. Here we introduce position anchoring in the alignment tool AlignMe as an aid to pairwise sequence alignment of membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of even a single anchor can dramatically improve the accuracy of the alignments, while maintaining the reproducibility and rigor of the overall alignment.

bioinformatics↗

bioRxiv2020

Global alignment and assessment of TRP channel transmembrane domain structures to explore functional mechanisms

The recent proliferation of published TRP channel structures provides a foundation for understanding the diverse functional properties of this important family of ion channel proteins. To facilitate mechanistic investigations, we constructed a structure-based alignment of the transmembrane domains of 120 TRP channel structures. Comparison of structures determined in the absence or presence of activating stimuli reveals similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available structures represent non-conducting states. Comparison of the ion selectivity filters towards the extracellular end of the pore supports existing hypotheses for mechanisms of ion selectivity. Also conserved to varying extents are hot spots for interactions with hydrophobic ligands, lipids and ions, as well as discrete alterations in helix conformations. This analysis therefore provides a framework for investigating the structural basis of TRP channel gating mechanisms and pharmacology, and, despite the large number of structures included, reveals the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function.

biophysics↗

bioRxiv2019

Structural Interpretation of Hydrogen-Deuterium Exchange with Maximum-Entropy Simulation Reweighting

Hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) is a widely applied biophysical technique that probes the structure and dynamics of biomolecules in native environments without the need for site-directed modifications or bio-orthogonal labels. The mechanistic interpretation of measured HDX data, however, is often qualitative and subjective, owing to a lack of quantitative methods to rigorously translate observed deuteration levels into atomistic structural information. To help address this problem, we have developed a methodology to generate structural ensembles that faithfully reproduce HDX-MS measurements. In this approach, an ensemble of protein conformations is first generated, typically using molecular dynamics simulations. A maximum-entropy bias is then applied post-hoc to the resulting ensemble, such that averaged peptide-deuteration levels, as predicted by an empirical model of a value called the protection factor, agree with target values within a given level of uncertainty. We evaluate this approach, referred to as HDX ensemble reweighting (HDXer), for artificial target data reflecting the two major conformational states of a binding protein. We demonstrate that the information provided by HDX-MS experiments, and by the model of exchange, are sufficient to recover correctly-weighted structural ensembles from simulations, even when the relevant conformations are observed rarely. Degrading the information content of the target data, e.g., by reducing sequence coverage or by averaging exchange levels over longer peptide segments, reduces the quantitative structural accuracy of the reweighted ensemble but still allows for useful, molecular-level insights into the distinctive structural features reflected by the target data. Finally, we describe a quantitative metric with which candidate structural ensembles can be ranked based on their correspondence with target data, or revealed to be inadequate. Thus, not only does HDXer facilitate a rigorous mechanistic interpretation of HDX-MS measurements, but it may also inform experimental design and further the development of empirical models of the HDX reaction.\n\nStatement of significanceHDX-MS experiments are a powerful approach for probing the conformational dynamics and mechanisms of proteins. However, the mechanistic implications of HDX-MS observations are frequently difficult to interpret, due to the limited spatial resolution of the technique as well as the lack of quantitative tools to translate measured data into structural information. To overcome these problems, we have developed a computational approach to construct structural ensembles that are maximally diverse while reproducing target experimental HDX-MS data within a given level of uncertainty. Using artificial test data, we demonstrate that the approach can correctly discern distinct structural ensembles reflected in the target data, and thereby facilitate statistically robust evaluations of competing mechanistic interpretations of HDX-MS experiments.

biophysics↗

bioRxiv2019

MemSTATS: A Benchmark Set of Membrane Protein Symmetries and Pseudo-Symmetries

In membrane proteins, symmetry and pseudo-symmetry often have functional or evolutionary implications. However, available symmetry detection methods have not been tested systematically on this class of proteins due to the lack of an appropriate benchmark set. Here we present MemSTATS, a publicly-available benchmark set of both quaternary and internal symmetries in membrane protein structures described in terms of order, repeated elements, and orientation of the axis with respect to the membrane plane. Moreover, using MemSTATS, we compare the performance of four widely-used symmetry detection algorithms and highlight specific challenges and areas for improvement in the future.

bioinformatics↗

bioRxiv2019

A structural model of the human serotonin transporter in an outward-occluded state

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

biophysics↗

bioRxiv2018

EncoMPASS: an Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry

Protein structure determination and prediction, active site detection, and protein sequence alignment techniques all exploit information about protein structure and structural relationships. For membrane proteins, however, there is no agreement among available online tools for highlighting and mapping such structural similarities. Moreover, no available resource provides a systematic overview of quaternary and internal symmetries, and their orientation with respect to the membrane, despite the fact that these properties can provide key insights into membrane protein function. To address these issues, we created the Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry (EncoMPASS), a database for relating integral membrane proteins of known structure from the points of view of sequence, structure, and symmetry. EncoMPASS is accessible at https://encompass.ninds.nih.gov and its contents can be easily downloaded. This allows the user not only to focus on specific systems, but also to study general properties of the structure and evolution of membrane proteins.\n\nHighlights-EncoMPASS relates and analyzes known structures of membrane proteins\n-Structure and sequence similarity is assessed through alignments and topology considerations, not clustering\n-Symmetry is detected based on CE-Symm and SymD using a multi-step procedure

bioinformatics↗

bioRxiv2018

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD (1), whereby the dynamics of the protein are minimally biased so as to reproduce the experimental data. Comparison of the magnitude of the biasing work required for different input structures permitted us to assign them to specific states of the transport cycle under each of the experimental conditions. In particular, this analysis showed that BetP adopts inward-facing conformations in the presence of excess sodium, and a mixture of states when betaine is added. These studies better delineate the major conformations adopted by BetP in its transport cycle, and therefore provide important insights into its mechanism. More broadly, we illustrate how integrative simulations can aid interpretation of ambiguous structural and spectroscopic data on membrane proteins.\n\nAbbreviations

biophysics↗

bioRxiv2018

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

biochemistry↗