Publications by authors named "Hassane S Mchaourab"

90 Publications

AlphaFold2 predicts the inward-facing conformation of the multidrug transporter LmrP.

Proteins 2021 May 11. Epub 2021 May 11.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

As part of the CASP competition, the protein structure prediction algorithm AlphaFold2 generated multiple models of the proton/drug antiporter LmrP. Previous distance restraints from double electron-electron resonance (DEER) spectroscopy, a technique which reports distance distributions between spin labels attached to proteins, suggest that one of the lower-ranked models may have captured a conformation that has so far eluded experimental structure determination. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/prot.26138DOI Listing
May 2021

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na in ion-coupled drug efflux.

J Biol Chem 2021 Jan 8;296:100262. Epub 2021 Jan 8.

Theoretical Molecular Biophysics Laboratory, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Electronic address:

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H-dependent activity likely stem either from a site elsewhere in the structure, or from H displacing Na under certain laboratory conditions, as has been noted for other Na-driven transport systems.
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http://dx.doi.org/10.1016/j.jbc.2021.100262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949106PMC
January 2021

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters.

J Mol Biol 2021 Mar 24:166959. Epub 2021 Mar 24.

Department of Molecular Physiology and Biophysics, Vanderbilt University, 747 Light Hall, 2215 Garland Avenue, Nashville, TN 37232, USA. Electronic address:

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.
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http://dx.doi.org/10.1016/j.jmb.2021.166959DOI Listing
March 2021

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na in ion-coupled drug efflux.

J Biol Chem 2021 Jan 5. Epub 2021 Jan 5.

National Institutes of Health, United States.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e. their drug-efflux activity is coupled to, and powered by, the uptake of ions down a pre-existing transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at resolutions better than 3 Å. Analysis of available crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.
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http://dx.doi.org/10.1074/jbc.RA120.016792DOI Listing
January 2021

Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy.

Sci Adv 2020 Dec 2;6(49). Epub 2020 Dec 2.

Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA.

Highly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human caveolin 1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N and C termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a model for how these highly stable membrane-embedded complexes are generated.
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http://dx.doi.org/10.1126/sciadv.abc6185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821874PMC
December 2020

The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters.

J Mol Biol 2020 09 26;432(20):5665-5680. Epub 2020 Aug 26.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA. Electronic address:

The prototypic multidrug (Mdr) transporter MdfA from Escherichia coli efflux chemically- dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (O) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (I) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distances measured by double electron-electron resonance (DEER) between pairs of spin labels in MdfA, reconstituted in nanodiscs, with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (O/I) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large-scale conformational changes. Rather, we identified a substrate-responsive lateral gate, which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access.
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http://dx.doi.org/10.1016/j.jmb.2020.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541688PMC
September 2020

ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter.

J Biol Chem 2020 10 20;295(43):14678-14685. Epub 2020 Aug 20.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA. Electronic address:

Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism. Here, we demonstrate the ATP-dependent, equilibrium binding of the cargo protein to the transmembrane domain (TMD) of a PCAT subsequent to the removal of the leader peptide by the PEP domain. Binding of the cargo protein to PCAT1 variants devoid of the PEP domain is detected through changes in the spectroscopic properties of fluorescent or spin label. Moreover, we find similar energetics of binding regardless of the presence of the leader peptide, suggesting that although the PEP domain serves for recognition and orientation, interaction with the TMD is the main contributor to the affinity. These findings are in direct contradiction with a recent study claiming that the TMD does not interact with the cargo protein; rather acting as a "Teflon-like" conduit across the bilayer (Kieuvongngam, V., Olinares, P. D. B., Palillo, A., Oldham, M. L., Chait, B. T., and Chen, J. (2020) Structural basis of substrate recognition by a polypeptide processing and secretion transporter. 9, e51492). A distinctive feature of the transport model emerging from our data invokes a stable complex between PCATs and their cargo proteins following processing of the leader peptide and prior to ATP-dependent alternating access that translocates the cargo protein to the extracellular side.
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http://dx.doi.org/10.1074/jbc.RA120.014934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586231PMC
October 2020

An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity.

Nat Struct Mol Biol 2020 09 27;27(9):829-835. Epub 2020 Jul 27.

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Brussels, Belgium.

Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.
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http://dx.doi.org/10.1038/s41594-020-0464-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951658PMC
September 2020

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl/H transport cycle.

Elife 2020 04 20;9. Epub 2020 Apr 20.

Department of Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, United States.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl for H). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H/Cl transport that reconciles existing data on all CLC-type proteins.
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http://dx.doi.org/10.7554/eLife.53479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253180PMC
April 2020

Supramolecular Approach to Electron Paramagnetic Resonance Distance Measurement of Spin-Labeled Proteins.

J Phys Chem B 2020 04 13;124(16):3291-3299. Epub 2020 Apr 13.

Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, United States.

We demonstrate a host-guest molecular recognition approach to advance double electron-electron resonance (DEER) distance measurements of spin-labeled proteins. We synthesized an iodoacetamide derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that could be doubly incorporated into T4 Lysozyme (T4L) by site-directed spin labeling with efficiency up to 50% per cysteine. The rigidity of the fused ring structure and absence of mobile methyl groups increase the spin echo dephasing time () at temperatures above 80 K. This enables DEER measurements of distances >4 nm in DZD-labeled T4L in glycerol/water at temperatures up to 150 K with increased sensitivity compared to that of a common spin label such as MTSL. Addition of β-cyclodextrin reduces the rotational correlation time of the label, slightly increases , and most importantly, narrows (and slightly lengthens) the interspin distance distributions. The distance distributions are in good agreement with simulated distance distributions obtained by rotamer libraries. These results provide a foundation for developing supramolecular recognition to facilitate long-distance DEER measurements at near physiological temperatures.
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http://dx.doi.org/10.1021/acs.jpcb.0c00743DOI Listing
April 2020

Sequence and structural determinants of ligand-dependent alternating access of a MATE transporter.

Proc Natl Acad Sci U S A 2020 03 19;117(9):4732-4740. Epub 2020 Feb 19.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.
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http://dx.doi.org/10.1073/pnas.1917139117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060674PMC
March 2020

Rapid Simulation of Unprocessed DEER Decay Data for Protein Fold Prediction.

Biophys J 2020 01 18;118(2):366-375. Epub 2019 Dec 18.

Department of Chemistry and Center for Structural Biology; Institut for Drug Discovery, Leipzig University, Leipzig, Germany. Electronic address:

Despite advances in sampling and scoring strategies, Monte Carlo modeling methods still struggle to accurately predict de novo the structures of large proteins, membrane proteins, or proteins of complex topologies. Previous approaches have addressed these shortcomings by leveraging sparse distance data gathered using site-directed spin labeling and electron paramagnetic resonance spectroscopy to improve protein structure prediction and refinement outcomes. However, existing computational implementations entail compromises between coarse-grained models of the spin label that lower the resolution and explicit models that lead to resource-intense simulations. These methods are further limited by their reliance on distance distributions, which are calculated from a primary refocused echo decay signal and contain uncertainties that may require manual refinement. Here, we addressed these challenges by developing RosettaDEER, a scoring method within the Rosetta software suite capable of simulating double electron-electron resonance spectroscopy decay traces and distance distributions between spin labels fast enough to fold proteins de novo. We demonstrate that the accuracy of resulting distance distributions match or exceed those generated by more computationally intensive methods. Moreover, decay traces generated from these distributions recapitulate intermolecular background coupling parameters even when the time window of data collection is truncated. As a result, RosettaDEER can discriminate between poorly folded and native-like models by using decay traces that cannot be accurately converted into distance distributions using regularized fitting approaches. Finally, using two challenging test cases, we demonstrate that RosettaDEER leverages these experimental data for protein fold prediction more effectively than previous methods. These benchmarking results confirm that RosettaDEER can effectively leverage sparse experimental data for a wide array of modeling applications built into the Rosetta software suite.
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http://dx.doi.org/10.1016/j.bpj.2019.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976798PMC
January 2020

Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels.

Sci Rep 2019 08 29;9(1):12528. Epub 2019 Aug 29.

Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.
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http://dx.doi.org/10.1038/s41598-019-48694-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715713PMC
August 2019

The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance.

J Biol Chem 2019 08 9;294(34):12807-12814. Epub 2019 Jul 9.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232

As a contributor to multidrug resistance, the family of multidrug and toxin extrusion (MATE) transporters couples the efflux of chemically dissimilar compounds to electrochemical ion gradients. Although divergent transport mechanisms have been proposed for these transporters, previous structural and functional analyses of members of the MATE subfamily DinF suggest that the N-terminal domain (NTD) supports substrate and ion binding. In this report, we investigated the relationship of ligand binding within the NTD to the drug resistance mechanism of the H-dependent MATE from the hyperthermophilic archaeon (PfMATE). To facilitate this study, we developed a cell growth assay in to characterize the resistance conferred by PfMATE to toxic concentrations of the antimicrobial compound rhodamine 6G. Expression of WT PfMATE promoted cell growth in the presence of drug, but amino acid substitutions of conserved NTD residues compromised drug resistance. Steady-state binding analysis with purified PfMATE indicated that substrate affinity was unperturbed in these NTD variants. However, exploiting Trp fluorescence as an intrinsic reporter of conformational changes, we found that these variants impaired formation of a unique H-stabilized structural intermediate. These results imply that disruption of H coupling is the origin of compromised toxin resistance in PfMATE variants. These findings support a model mechanism wherein the NTD mediates allosteric coupling to ion gradients through conformational changes to drive substrate transport in PfMATE. Furthermore, the results provide evidence for diverging transport mechanisms within a prokaryotic MATE subfamily.
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http://dx.doi.org/10.1074/jbc.RA119.009195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709631PMC
August 2019

Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors.

Science 2019 05;364(6441):689-692

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA.

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5'-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.
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http://dx.doi.org/10.1126/science.aav9406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890515PMC
May 2019

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in .

Proc Natl Acad Sci U S A 2019 02 12;116(9):3853-3862. Epub 2019 Feb 12.

Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35233;

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.
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http://dx.doi.org/10.1073/pnas.1816247116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397532PMC
February 2019

Transgenic zebrafish models reveal distinct molecular mechanisms for cataract-linked αA-crystallin mutants.

PLoS One 2018 26;13(11):e0207540. Epub 2018 Nov 26.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, United States of America.

Mutations in the small heat shock proteins α-crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of αA- and αB-crystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous α-crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human γD-crystallin mutant. We found that the R49C but not the R116C mutant of αA-crystallin drove aggregation of γD-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of αA-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207540PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261105PMC
April 2019

Structural Basis of H-Dependent Conformational Change in a Bacterial MATE Transporter.

Structure 2019 02 15;27(2):293-301.e3. Epub 2018 Nov 15.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds using a Na or H gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H-coupled MATE transporters.
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http://dx.doi.org/10.1016/j.str.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935464PMC
February 2019

Movement of the RecG Motor Domain upon DNA Binding Is Required for Efficient Fork Reversal.

Int J Mol Sci 2018 Oct 6;19(10). Epub 2018 Oct 6.

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232, USA.

RecG catalyzes reversal of stalled replication forks in response to replication stress in bacteria. The protein contains a fork recognition ("wedge") domain that binds branched DNA and a superfamily II (SF2) ATPase motor that drives translocation on double-stranded (ds)DNA. The mechanism by which the wedge and motor domains collaborate to catalyze fork reversal in RecG and analogous eukaryotic fork remodelers is unknown. Here, we used electron paramagnetic resonance (EPR) spectroscopy to probe conformational changes between the wedge and ATPase domains in response to fork DNA binding by RecG. Upon binding DNA, the ATPase-C lobe moves away from both the wedge and ATPase-N domains. This conformational change is consistent with a model of RecG fully engaged with a DNA fork substrate constructed from a crystal structure of RecG bound to a DNA junction together with recent cryo-electron microscopy (EM) structures of chromatin remodelers in complex with dsDNA. We show by mutational analysis that a conserved loop within the translocation in RecG (TRG) motif that was unstructured in the RecG crystal structure is essential for fork reversal and DNA-dependent conformational changes. Together, this work helps provide a more coherent model of fork binding and remodeling by RecG and related eukaryotic enzymes.
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http://dx.doi.org/10.3390/ijms19103049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213257PMC
October 2018

Confidence Analysis of DEER Data and Its Structural Interpretation with Ensemble-Biased Metadynamics.

Biophys J 2018 10 16;115(7):1200-1216. Epub 2018 Aug 16.

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee. Electronic address:

Given its ability to measure multicomponent distance distributions between electron-spin probes, double electron-electron resonance (DEER) spectroscopy has become a leading technique to assess the structural dynamics of biomolecules. However, methodologies to evaluate the statistical error of these distributions are not standard, often hampering a rigorous interpretation of the experimental results. Distance distributions are often determined from the experimental DEER data through a mathematical method known as Tikhonov regularization, but this approach makes rigorous error estimates difficult. Here, we build upon an alternative, model-based approach in which the distance probability distribution is represented as a sum of Gaussian components, and use propagation of errors to calculate an associated confidence band. Our approach considers all sources of uncertainty, including the experimental noise, the uncertainty in the fitted background signal, and the limited time span of the data collection. The resulting confidence band reveals the most and least reliable features of the probability distribution, thereby informing the structural interpretation of DEER experiments. To facilitate this interpretation, we also generalize the molecular simulation method known as ensemble-biased metadynamics (EBMetaD). This method, originally designed to generate maximal-entropy structural ensembles consistent with one or more probability distributions, now also accounts for the uncertainty in those target distributions exactly as dictated by their confidence bands. After careful benchmarks, we demonstrate the proposed techniques using DEER results from spin-labeled T4 lysozyme.
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http://dx.doi.org/10.1016/j.bpj.2018.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170522PMC
October 2018

Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity.

Structure 2018 08 5;26(8):1116-1126.e4. Epub 2018 Jul 5.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville 37232, TN, USA. Electronic address:

Small heat-shock proteins (sHSPs) are molecular chaperones that bind partially and globally unfolded states of their client proteins. Previously, we discovered that the archaeal Hsp16.5, which forms ordered and symmetric 24-subunit oligomers, can be engineered to transition to an ordered and symmetric 48-subunit oligomer by insertion of a peptide from human HspB1 (Hsp27). Here, we uncovered the existence of an array of oligomeric states (30-38 subunits) that can be populated as a consequence of altering the sequence and length of the inserted peptide. Polydisperse Hsp16.5 oligomers displayed higher affinity to a model client protein consistent with a general mechanism for recognition and binding that involves increased access of the hydrophobic N-terminal region. Our findings, which integrate structural and functional analyses from evolutionarily distant sHSPs, support a model wherein the modular architecture of these proteins encodes motifs of oligomer polydispersity, dissociation, and expansion to achieve functional diversity and regulation.
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http://dx.doi.org/10.1016/j.str.2018.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563326PMC
August 2018

Sodium and proton coupling in the conformational cycle of a MATE antiporter from .

Proc Natl Acad Sci U S A 2018 07 18;115(27):E6182-E6190. Epub 2018 Jun 18.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232

Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from (NorM-Vc), a MATE transporter proposed to be coupled to both Na and H gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na, H, or the substrate doxorubicin. The Na- and H-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na- and H-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H in the doxorubicin release mechanism.
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http://dx.doi.org/10.1073/pnas.1802417115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142240PMC
July 2018

Mechanism of NMDA receptor channel block by MK-801 and memantine.

Nature 2018 04 18;556(7702):515-519. Epub 2018 Apr 18.

Vollum Institute, Oregon Health & Science University, Portland, OR, USA.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.
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http://dx.doi.org/10.1038/s41586-018-0039-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962351PMC
April 2018

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Proc Natl Acad Sci U S A 2018 03 5;115(12):E2742-E2751. Epub 2018 Mar 5.

Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90096;

Sodium-dependent transporters couple the flow of Na ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na-dependent sugar transporter from (vSGLT) has previously been reported, but structural details on additional conformations and on how Na and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na, vSGLT favors an inward-facing conformation. Upon binding both Na and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.
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http://dx.doi.org/10.1073/pnas.1718451115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866573PMC
March 2018

Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart.

J Biol Chem 2018 01 21;293(2):740-753. Epub 2017 Nov 21.

From the Departments of Molecular Physiology and Biophysics and

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity , the functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, α and α We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in α than α mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.
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http://dx.doi.org/10.1074/jbc.M117.808634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767876PMC
January 2018

Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters.

Structure 2017 08 14;25(8):1264-1274.e3. Epub 2017 Jul 14.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover.
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http://dx.doi.org/10.1016/j.str.2017.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600498PMC
August 2017

The growing world of small heat shock proteins: from structure to functions.

Cell Stress Chaperones 2017 07 31;22(4):601-611. Epub 2017 Mar 31.

Laboratory of Cell & Developmental Genetics, IBIS, and Department of Molecular Biology, Medical Biochemistry and Pathology, Medical School, Université Laval, Québec (Qc), G1V 0A6, Canada.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).
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http://dx.doi.org/10.1007/s12192-017-0787-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465036PMC
July 2017

Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein.

Nature 2017 03 13;543(7647):738-741. Epub 2017 Mar 13.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.

ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD). Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron-electron resonance and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.
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http://dx.doi.org/10.1038/nature21414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558441PMC
March 2017

Alternating access mechanisms of LeuT-fold transporters: trailblazing towards the promised energy landscapes.

Curr Opin Struct Biol 2017 08 30;45:100-108. Epub 2016 Dec 30.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States. Electronic address:

Secondary active transporters couple the uphill translocation of substrates to electrochemical ion gradients. Transporter conformational motion, generically referred to as alternating access, enables a central ligand binding site to change its orientation relative to the membrane. Here we review themes of alternating access and the transduction of ion gradient energy to power this process in the LeuT-fold class of transporters where crystallographic, computational and spectroscopic approaches have converged to yield detailed models of transport cycles. Specifically, we compare findings for the Na-coupled amino acid transporter LeuT and the Na-coupled hydantoin transporter Mhp1. Although these studies have illuminated multiple aspects of transporter structures and dynamics, a number of questions remain unresolved that so far hinder understanding transport mechanisms in an energy landscape perspective.
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http://dx.doi.org/10.1016/j.sbi.2016.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491374PMC
August 2017

Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

J Biol Chem 2016 Dec 21;291(49):25387-25397. Epub 2016 Oct 21.

From the Departments of Molecular Physiology and Biophysics and

The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different γD-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of αA-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that αA-crystallin may not be efficient to inhibit aggregation of lens γ-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.
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http://dx.doi.org/10.1074/jbc.M116.749606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207241PMC
December 2016