Biology subjects

Govaerts, C.

Publications and source records attributed to Govaerts, C..

7 recordsLinked to original sources

bioRxiv2026

Local conformational plasticity underlies ligand recognition and 1 proton coupling in MFS multidrug transporters

Polyspecific substrate recognition drives multidrug efflux and antibiotic resistance, yet its molecular basis remains unclear. Here, we use HDX-MS to compare ligand-dependent local dynamics in three multidrug efflux pumps: NorA, QacA and LmrP. In the apo state, all three display high flexibility in specific transmembrane helices, unlike homologous transporters with narrow substrate profiles. Substrate binding remodels these flexible regions, but in a transporter-specific manner, revealing divergent local adaptations within a conserved fold. In LmrP, protonation-mimicking mutation of a conserved acidic residue recapitulates the substrate-induced dynamic changes, supporting a model in which transmembrane helix flexibility couples protonation and substrate binding to the conformational changes required for transport. Together, our study identifies local plasticity beyond what is captured by static high-resolution structures, as an overlooked feature of polyspecific ligand recognition.

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

Nanobody-Driven Stabilization Synergistically Rescues F508del-CFTR and Reveals an Alternative Active State of the Channel

Defects in protein trafficking underlie many genetic diseases, including cystic fibrosis (CF), where the predominant F508del mutation destabilizes the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to its degradation. To provide a protein-specific chaperone, we used lipid nanoparticles to deliver mRNA encoding T2a, a nanobody that thermally stabilizes CFTR via high-affinity binding to nucleotide-binding domain 1 (NBD1). When combined with clinically-approved correctors, T2a considerably improved F508del-CFTR maturation, plasma membrane expression, and channel activity. Single-channel recordings revealed that nanobody binding sustained channel activity by promoting both full open and sub-conductance gating states and protecting F508del-CFTR against thermal deactivation. Because T2a binding to NBD1 prevents the ATP-dependent NBD1-NBD2 association that drives canonical channel opening, the observation of channel activity in the presence of the nanobody suggested adoption of a novel open-channel CFTR structure. This inference was confirmed by cryo-EM analyses of CFTR in the presence of T2a, which revealed a novel open-channel conformation in which NBD1 binds to an alternative site on CFTR incompatible with NBD dimerization. Our findings establish a new paradigm to correct protein trafficking by stabilizing misfolded domains with targeted nanobodies and demonstrate a broadly applicable framework to treat CF and related protein misfolding diseases.

biochemistry↗

bioRxiv2025

Antibiotics-induced conformational heterogeneity of a multidrug transporter revealed by single-molecule FRET

Multidrug transporters are membrane proteins that can transport an ensemble of structurally dissimilar compounds and contribute to bacterial multidrug resistance (MDR) by exporting different antibiotics from the cell. However, whether they transport different substrates through a common mechanism or via distinct substrate-dependent mechanisms remains unclear. In this work, we used single-molecule Forster resonance energy transfer (smFRET) to measure time-resolved conformational dynamics of LmrP, a multidrug transporter of the Major Facilitator Superfamily (MFS). We present high-resolution conformational landscapes of LmrP in the presence of different antibiotics. Through multi-parameter Hidden Markov Modeling (mpH2MM), we uncovered transient states and quantified their sub-millisecond interconversion kinetics. We observed antibiotic-dependent heterogeneity in the conformational landscape, both in accessible states and in interconversion rates. Notably, poorly or non-transported antibiotics slow down transition kinetics, pointing to rapid state interconversion as a driver of efficient transport. This suggests that MFS MDR transporters bind and export structurally dissimilar antibiotics by relying on an array of underlying conformational states with ligand-dictated interconversion rates. This work provides novel insights into the mechanism of MDR transporters and advocates for combined structure/dynamics-based drug design when targeting their function.

biophysics↗

bioRxiv2024

A Cell-Permeable Nanobody to Restore F508del Cystic Fibrosis Transmembrane Conductance Regulator Activity

Nanobodies have gained considerable attention as particularly promising biopharmaceuticals. However, nanobody-based modalities are currently limited to extracellular targets due to a lack of efficient delivery methods required to reach targets inside cells. In this study, we introduce cell-permeable nanobodies for targeting a disease-relevant intracellular protein, namely the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with the most common cystic fibrosis (CF)-causing mutation F508del. We employ cell-penetrating peptides (CPPs) to deliver a CFTR-binding nanobody (NB1) that stabilizes misfolded F508del-CFTR and prevents its degradation to restore its function. Our data show that conjugation of a disulfide-linked CPP in combination with a cell-surface anchored CPP-additive enables intracellular delivery of NB1 into CF bronchial epithelial cells, which promotes maturation and trafficking of F508del-CFTR protein to the apical cell membrane. Furthermore, we demonstrate that the cell-permeable nanobody restores CFTR chloride channel function, which can be further enhanced by the clinically approved small molecule CFTR potentiator ivacaftor. This study highlights the use of cell-permeable nanobodies for modulation of protein function and illustrates their therapeutic potential as next-generation biopharmaceuticals for intracellular delivery and targeting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/591242v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@45d357org.highwire.dtl.DTLVardef@362e56org.highwire.dtl.DTLVardef@11c4dc2org.highwire.dtl.DTLVardef@13512b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Forster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically between the 3 states (< 10 ms dwell time) at the extracellular side. When the Hoechst 33342 model substrate is added, inward conformational interconversions are greatly accelerated, coupled to an overall outward conformational halting, consistently with efficient proton exchange with the extracellular environment. Roxithromycin substrate binding did not halt but shift conformational interconversions from one pair of states to another. Substrate dependent structural heterogeneity is indicative of a general mechanism by which MFS transporters can efficiently transport a variety of substrates, and advocates for combined structure/dynamics-based drug design when targeting MDR transporters. BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Forster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.

biophysics↗

bioRxiv2019

A functionally conserved mechanism of modulation via a vestibule site in pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) belong to a class of ion channels involved in fast synaptic signaling in the central and peripheral nervous systems. Molecules acting as allosteric modulators target binding sites that are remote from the neurotransmitter binding site, but functionally affect coupling of ligand binding to channel opening. Here, we investigated an allosteric binding site in the ion channel vestibule, which has converged from a series of studies on prokaryote and eukaryote channel homologs. We discovered single domain antibodies, called nanobodies, which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote channel ELIC bound either to a positive (PAM) or a negative (NAM) allosteric modulator. We extrapolate the functional importance of the vestibule binding site to eukaryote ion channels, suggesting a conserved mechanism of allosteric modulation. This work identifies key elements of allosteric binding sites and extends drug design possibilities in pLGICs using nanobodies.

neuroscience↗