Biology subjects

Quick, M.

Publications and source records attributed to Quick, M..

12 recordsLinked to original sources

bioRxiv2024

Impact of anionic lipids on the energy landscape of conformational transition in anion exchanger 1 (AE1)

Anion Exchanger 1 (AE1) is an elevator-type membrane transporter that plays a key role in erythrocytes by mediating the exchange of chloride and bicarbonate ions across the membrane, thus participating in acid-base homeostasis. While previous studies have provided structural insights into AE1 and its substrate binding, the conformational transitions and the role of lipid interactions remain elusive. In this study, we utilized cryo-electron microscopy (cryo-EM) to resolve three high resolution structures of distinct conformational states of AE1: two inward-facing (IF1 and IF2) and one outward-facing (OF). Furthermore, uptake assay revealed the modulatory effect of phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on AE1. Molecular dynamics (MD) simulations were conducted on these structures to capture anion binding and determine the anion binding sites in AE1. We then used a combination of advanced enhanced sampling techniques together with system-specific collective variables to study the OF{lrdoublearrow}IF transition in AE1 and provided refined pathways for the process in three different systems: apo, HCO3- -bound, and an AE1 system in which cryo-EM-determined interfacial PIP2 lipids had been removed. The refined pathways were then used to calculate the free energy of the OF{lrdoublearrow}IF transition in AE1 under different conditions. The energies show how substrate binding reduces the transition barrier and, therefore, facilitates the transport. Furthermore, they clearly capture the inhibitory effect of PIP2 lipids at the dimer interface. Furthermore, the simulation results provide a molecular mechanism for this inhibitory effect. These results provide a molecular-level understanding of the mechanistic basis for ion transport in AE1 and the regulatory role of PIP2 on its function.

biophysics↗

bioRxiv2024

Identification of the drug/metabolite transporter 1 as a marker of quinine resistance in a NF54xCam3.II P. falciparum genetic cross.

The genetic basis of Plasmodium falciparum resistance to quinine (QN), a drug used to treat severe malaria, has long been enigmatic. To gain further insight, we used FRG-NOD human liver-chimeric mice to conduct a P. falciparum genetic cross between QN-resistant (Cam3.II) and QN-sensitive (NF54) parasites, which also differ in their susceptibility to chloroquine (CQ). By applying different selective conditions to progeny pools prior to cloning, we recovered 120 unique recombinant progeny. Drug profiling and quantitative trait loci analyses of the progeny revealed predominant peaks on chromosomes 7 and 12 associated with CQ and QN resistance, that is consistent with a multifactorial mechanism of resistance for these compounds. CQ and monodesethyl-CQ (md-CQ) resistance mapped to a chromosome 7 region harboring pfcrt as expected. However, for QN, resistance mapped to a dominant chromosome 7 peak centered 295 kb downstream of pfcrt, with pfcrt showing a smaller peak. We identified the drug/metabolite transporter 1 (DMT1) as the top chromosome 7 candidate due to its structural similarity to PfCRT and proximity to the peak. Deleting DMT1 in QN-resistant Cam3.II parasites significantly sensitized the parasite to QN but not to the other drugs tested, suggesting that DMT1 mediates QN response specifically. We localized DMT1 to structures associated with vesicular trafficking, as well as the parasitophorous vacuolar membrane, lipid bodies, and the digestive vacuole. We also observed that mutant DMT1 transports more QN than the wild-type isoform in vitro. Gene editing confirmed an additional role for mutant PfCRT in mediating QN resistance. In addition, we identified an ATP-dependent zinc metalloprotease (FtsH1) as one of the top candidates in the chromosome 12 locus and confirmed its role as a potential mediator of QN resistance and a modulator of md-CQ resistance using CRISPR/Cas9 SNP-edited lines. Interestingly, this chromosome 12 region mapped to resistance to both CQ and QN and was preferentially co-inherited with pfcrt. Our study demonstrates that DMT1 is a novel marker of QN resistance and that a new chromosome 12 locus associates with CQ and QN response, with ftsh1 as a potential candidate, suggesting these genes in addition to pfcrt should be genotyped in surveillance and clinical settings.

genetics↗

bioRxiv2024

Alternating Access of a Neurotransmitter:Sodium Symporter Bacterial Homolog Determined from AlphaFold2 Ensembles and DEER Spectroscopy

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understands the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a novel computational/experimental methodology to illuminate the energy landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movement of multiple structural motifs that underpins MhsTs conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlight the profound effect of the environment on the energetics of conformational changes. Through experimentally-derived selection of collective variables, we present a model of ion and substrate powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsTs function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces a novel approach that can be applied across a diverse spectrum of transporters to describe their energy landscapes. Significance StatementThe neurotransmitter:sodium symporter (NSS) family plays a crucial role in neurotransmitter reuptake, a sodium-dependent process that transports neurotransmitters from the synapse back into the neuron. This study investigates the bacterial tryptophan transporter MhsT, a homolog of human NSSs, using the deep learning method AlphaFold2 in conjunction with double electron-electron resonance spectroscopy. This combined approach enables us to map the energy landscape that dictates the conformational shifts crucial for MhsTs function. Furthermore, we reveal how the environment modulates the transporters dynamics. From our research, we develop a model of MhsT transport that highlights the extent of mechanistic conservation across the NSS family. Additionally, we introduce a comprehensive framework for exploring the energetic landscapes of transporters, effectively integrating computational and experimental methods.

biophysics↗

bioRxiv2023

Structural and molecular basis of choline uptake into the brain by FLVCR2

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 [A] resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

biochemistry↗

bioRxiv2023

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration

Parkinsons disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1{beta} as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

neuroscience↗

bioRxiv2023

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

genomics↗

bioRxiv2022

Theta phase specific modulation of hippocampal memory neurons

I.The theta rhythm, a quasi-periodic 4-10 Hz oscillation, is observed during memory processing in the hippocampus, with different phases of theta hypothesized to separate independent streams of information related to the encoding and recall of memories. At the cellular level, the discovery of hippocampal memory cells (engram neurons), as well as the modulation of memory recall through optogenetic activation of these cells, has provided evidence that certain memories are stored, in part, in a sparse ensemble of neurons in the hippocampus. In previous research, however, engram reactivation has been carried out using open loop stimulation at fixed frequencies; the relationship between engram neuron reactivation and ongoing network oscillations has not been taken into consideration. To address this concern, we implemented a closed-loop reactivation of engram neurons that enabled phase-specific stimulation relative to theta oscillations in the local field potential. Using this real-time approach, we tested the impact of activating engram neurons during the peak (encoding phase) and trough (recall phase) of theta oscillations. Consistent with previously hypothesized functions of theta oscillations in memory function, we show that stimulating engram neurons at the trough of theta is more effective in eliciting behavioral recall than either fixed frequency stimulation or stimulation at the peak of theta. Moreover, phase-specific trough stimulation is accompanied by an increase in the coupling between gamma and theta oscillations in CA1 hippocampus. Oure results provide a causal link between phase-specific activation of engram cells and the behavioral expression of memory.

neuroscience↗

bioRxiv2021

Insight into the mechanism of H+-coupled nucleobase transport

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 [A] resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

biochemistry↗

bioRxiv2020

Developing Small Molecules that Inhibit K-Ras/GTP Binding Based on New Affinity Measurements

RAS genes encode small GTPases essential for proliferation, differentiation, and survival of mammalian cells. RAS gene mutations are associated with approximately 30% of all human cancers. However, based on measurements reported three decades ago of Ras protein affinities to GTP in the 10-20 picomolar range, it has been accepted in the scientific and medical communities that Ras proteins are undruggable targets. Here, we report MicroScale Thermophoresis and scintillation proximity assay measurements of the affinity of K-Ras and several K-Ras mutants for GTP in the range of 200 nanomolar, a 10,000-fold difference from that previously reported, and the identification of over 400 small molecules that block GTP binding to K-Ras. Focusing on two of those molecules, we report small molecule inhibition of Ras downstream signaling and cellular proliferation in human pancreatic and non-small cell lung cancer cells expressing wild type and K-Ras G12C, G12D and G12S, and N-Ras Q61K mutants.

cancer biology↗

bioRxiv2020

Role of transmembrane helix 6 in substrate recognition of the amino acid transporter MhsT

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C){Phi}G motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

biochemistry↗

bioRxiv2019

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

biophysics↗

bioRxiv2017

Structure-based analysis of CysZ-mediated cellular uptake of sulfate

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. Current understanding of the cellular delivery of SO42- at the molecular level is limited however. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and uptake experiments in cells and proteoliposomes, and single-channel conductance measurements, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ properties differ markedly from those of known transporters and ion channels. The structures display a hitherto unknown fold with dual topology, assembling in CysZ from Pseudomonas denitrificans as a trimer of antiparallel dimers in the membrane. CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

biophysics↗