RESUMEN
Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Sistemas Toxina-Antitoxina , Sistemas Toxina-Antitoxina/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Escherichia coli/virología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Bacteriófago lambda/genética , Bacteriófago lambda/fisiología , Bacteriófago lambda/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/fisiología , Antitoxinas/metabolismo , Antitoxinas/genética , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/genéticaRESUMEN
Acinetobacter baumannii is a nosocomial pathogen highly resistant to environmental changes and antimicrobial treatments. Regulation of cellular motility and biofilm formation is important for its virulence, although it is poorly described at the molecular level. It has been previously reported that Acinetobacter genus specifically produces a small positively charged metabolite, polyamine 1,3-diaminopropane, that has been associated with cell motility and virulence. Here we show that A. baumannii encodes novel acetyltransferase, Dpa, that acetylates 1,3-diaminopropane, directly affecting the bacterium motility. Expression of dpa increases in bacteria that form pellicle and adhere to eukaryotic cells as compared to planktonic bacterial cells, suggesting that cell motility is linked to the pool of non-modified 1,3-diaminopropane. Indeed, deletion of dpa hinders biofilm formation and increases twitching motion confirming the impact of balancing the levels of 1,3-diaminopropane on cell motility. The crystal structure of Dpa reveals topological and functional differences from other bacterial polyamine acetyltransferases, adopting a ß-swapped quaternary arrangement similar to that of eukaryotic polyamine acetyltransferases with a central size exclusion channel that sieves through the cellular polyamine pool. The structure of catalytically impaired DpaY128F in complex with the reaction product shows that binding and orientation of the polyamine substrates are conserved between different polyamine-acetyltransferases.
Asunto(s)
Acinetobacter baumannii , Acinetobacter baumannii/genética , Acetiltransferasas/genética , Poliaminas , BiopelículasRESUMEN
Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.
RESUMEN
Transporters regulate trafficking through the biological membrane of living cells and organelles. Therefore, these proteins play an important role in key cellular processes. Obtaining a molecular-level description of the mechanism of transporters is highly desirable to understand and modulate such processes. Different challenges currently complicate this effort, mostly due to transporters' intrinsic properties. They are dynamic and often averse to in vitro characterization. The crossing of the membrane via a transporter depends on both global and local structural changes that will enable substrate binding from one side of the membrane and release on the other. Dedicated approaches are required to monitor these dynamic changes, ideally within the complex membrane environment. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool to understand transporters' mechanism. This mini-review aims to offer to the reader an overview of the field of HDX-MS applied to transporters. It first summarizes the current workflow for HDX-MS measurements on transporters. It then provides illustrative examples on the molecular insights that are accessible thanks to the technique; following conformational transitions between different states, observing structural changes upon ligand binding and finally understanding the role of lipid-protein interactions.
Asunto(s)
Medición de Intercambio de Deuterio , Hidrógeno , Hidrógeno/química , Deuterio , Medición de Intercambio de Deuterio/métodos , Conformación Proteica , Espectrometría de Masas/métodos , Proteínas de Transporte de MembranaRESUMEN
Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity.
Asunto(s)
Bacteriófagos , Proteínas de la Cápside , Escherichia coli , Inmunidad Innata , Animales , Antitoxinas/inmunología , Bacteriófagos/inmunología , Proteínas de la Cápside/inmunología , Escherichia coli/inmunología , Escherichia coli/virología , Eucariontes/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/inmunologíaRESUMEN
Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Proteínas Arqueales/metabolismo , Membrana Celular/metabolismo , Medición de Intercambio de Deuterio , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG/genética , Proteínas Arqueales/genética , Transporte Biológico , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Espectrometría de Masas , Mutación , Unión Proteica , Imagen Individual de MoléculaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the ß-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/aislamiento & purificación , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Desplegamiento ProteicoRESUMEN
Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid-protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid-protein interactions in the mechanism of membrane proteins.
Asunto(s)
Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Humanos , Unión Proteica/fisiologíaRESUMEN
Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.
Asunto(s)
Proteínas de Escherichia coli/química , Glucosa/química , Protones , Simportadores/química , Xilosa/química , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucosa/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Cinética , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Simportadores/antagonistas & inhibidores , Simportadores/genética , Simportadores/metabolismo , Termodinámica , Xilosa/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Lactococcus lactis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilgliceroles/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Lactococcus lactis/química , Ligandos , Proteínas de Transporte de Membrana/química , Simulación de Dinámica Molecular , Fosfatidilgliceroles/química , Conformación Proteica , Especificidad por SustratoRESUMEN
The expression and downstream purification of membrane proteins is the prerequisite for biophysical and structural studies of this major source of therapeutic targets. The gram-positive bacterium Lactococcus lactis is an attractive option for heterologous membrane protein expression and purification thanks to advantageous characteristics such as mild proteolytic activity and small genome size. Vectors designed for gene transcription under the control of inducible promoters are readily available. Specifically, the tightly regulated nisin-inducible gene expression system (NICE) allows to fine-tune the overexpression of different gene products. The expressed protein engineered with a suitable tag can be readily detected and purified from crude membrane extracts. The purpose of this protocol chapter is to detail the procedures of cloning, expression, isolation of the membrane vesicles, and affinity purification of a membrane protein of interest in L. lactis.
Asunto(s)
Clonación Molecular/métodos , Lactococcus lactis , Proteínas de la Membrana , Conformación Proteica , Proteínas Recombinantes , Membrana Celular/química , Membrana Celular/metabolismo , Fraccionamiento Químico/métodos , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Lactococcus lactis/química , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Nisina/química , Nisina/genética , Nisina/metabolismo , Organismos Modificados Genéticamente , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia/métodos , Transformación BacterianaRESUMEN
Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research. They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High-resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX-MS as it has matured into an essential tool for membrane protein structural biologists.
Asunto(s)
Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteínas de la Membrana/química , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Biological membranes define the boundaries of cells and are composed primarily of phospholipids and membrane proteins. It has become increasingly evident that direct interactions of membrane proteins with their surrounding lipids play key roles in regulating both protein conformations and function. However, the exact nature and structural consequences of these interactions remain difficult to track at the molecular level. Here, we present a protocol that specifically addresses this challenge. First, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of membrane proteins incorporated into nanodiscs of controlled lipid composition is used to obtain information on the lipid species that are involved in modulating the conformational changes in the membrane protein. Then molecular dynamics (MD) simulations in lipid bilayers are used to pinpoint likely lipid-protein interactions, which can be tested experimentally using HDX-MS. By bringing together the MD predictions with the conformational readouts from HDX-MS, we have uncovered key lipid-protein interactions implicated in stabilizing important functional conformations. This protocol can be applied to virtually any integral membrane protein amenable to classic biophysical studies and for which a near-atomic-resolution structure or homology model is available. This protocol takes ~4 d to complete, excluding the time for data analysis and MD simulations, which depends on the size of the protein under investigation.
Asunto(s)
Membrana Celular/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Medición de Intercambio de Deuterio , Espectrometría de Masas , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.
Asunto(s)
Complejo del Señalosoma COP9/ultraestructura , Proteína NEDD8/ultraestructura , Péptido Hidrolasas/ultraestructura , Ubiquitina-Proteína Ligasas/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Complejo del Señalosoma COP9/aislamiento & purificación , Complejo del Señalosoma COP9/metabolismo , Microscopía por Crioelectrón , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espectrometría de Masas , Proteína NEDD8/aislamiento & purificación , Proteína NEDD8/metabolismo , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Células Sf9 , Ubiquitina-Proteína Ligasas/aislamiento & purificación , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
SUMMARY: Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, analysis of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data analysis software, allowing the rapid analysis and visualization of data from differential HDX-MS. AVAILABILITY AND IMPLEMENTATION: Deuteros is open-source and can be downloaded from https://github.com/andymlau/Deuteros, under the Apache 2.0 license. Written in MATLAB and supported on both Windows and MacOS. Requires the MATLAB runtime library. According to the Wellcome Trust and UK research councils' Common Principles on Data Policy on data, software and materials management and sharing, all data supporting this study will be openly available from the software repository.
Asunto(s)
Medición de Intercambio de Deuterio , Programas Informáticos , Hidrógeno , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Espectrometría de Masas , ProteínasRESUMEN
Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.
Asunto(s)
Espectrometría de Masas , Complejos Multiproteicos/química , Proteínas/química , Espectrometría de Movilidad Iónica , Ligandos , Simulación de Dinámica Molecular , Complejos Multiproteicos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas/metabolismoRESUMEN
Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.
Asunto(s)
Proteínas de Escherichia coli/química , Lípidos de la Membrana/química , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Conformación Proteica , Membrana Celular/metabolismo , Medición de Intercambio de Deuterio , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Simulación de Dinámica Molecular , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Unión Proteica , Simportadores/química , Simportadores/metabolismoRESUMEN
The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene-maleic acid lipid particle (SMALP) technology can be coupled with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.
Asunto(s)
Lípidos/química , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ADN/metabolismo , Medición de Intercambio de Deuterio , Endopeptidasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/química , Conformación ProteicaRESUMEN
Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers.
Asunto(s)
Proteínas Bacterianas/química , Lactococcus lactis , Lípidos de la Membrana/química , Proteínas de Transporte de Membrana/química , Cardiolipinas/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad ProteicaRESUMEN
Multidrug antiporters of the major facilitator superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we used extensive double electron-electron resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from Lactococcus lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations, depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.