RESUMO
Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.
RESUMO
The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.
Assuntos
Antibacterianos , Bacitracina , Antibacterianos/química , Testes de Sensibilidade Microbiana , BactériasRESUMO
The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here, we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a triazole-linked bullvalene core, exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding d-Ala-d-Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, implying the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Enterococos Resistentes à Vancomicina , Vancomicina/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (â¼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of â¼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Ligação Proteica , Espectrometria de Massas , PolissacarídeosRESUMO
Integral membrane proteins are involved in a plethora of biological processes including cellular signalling, molecular transport, and catalysis. Many of these functions are mediated by non-covalent interactions with other proteins, substrates, metabolites, and surrounding lipids. Uncovering such interactions and deciphering their effect on protein activity is essential for understanding the regulatory mechanisms underlying integral membrane protein function. However, the detection of such dynamic complexes has proven to be challenging using traditional approaches in structural biology. Native mass spectrometry has emerged as a powerful technique for the structural characterisation of membrane proteins and their complexes, enabling the detection and identification of protein-binding partners. In this review, we discuss recent native mass spectrometry-based studies that have characterised non-covalent interactions of membrane proteins in the presence of detergents or membrane mimetics. We additionally highlight recent progress towards the study of membrane proteins within native membranes and provide our perspective on how these could be combined with recent developments in instrumentation to investigate increasingly complex biomolecular systems.
Assuntos
Proteínas de Membrana , Proteínas de Membrana/metabolismo , Espectrometria de Massas/métodos , Ligação ProteicaRESUMO
The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) is essential for Gram-negative bacteria OM barrier function and for maintaining its cell integrity. As such, comprehensive information about its biosynthesis and translocation represents a successful strategy for the development of antibacterial drugs. LPS is a complex glycolipid, and probing its interactions with LPS transport (Lpt) proteins has been extremely challenging. However, mass spectrometry (MS) techniques have recently catalyzed tremendous advancements in the characterization of LPS transport (Lpt) proteins and probed associated conformational dynamics upon substrate binding. Here, we describe the application of MS methods to study the dynamics of LPS translocon LptDE in the presence of natural substrates and inhibitors.
Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Antibacterianos , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Proteínas de Escherichia coli/metabolismo , Glicolipídeos , Lipopolissacarídeos/química , Espectrometria de MassasRESUMO
Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism.
Assuntos
Escherichia coli , Homosserina , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Homosserina/análogos & derivados , Homosserina/análise , Homosserina/metabolismo , Lactonas , Simulação de Acoplamento Molecular , Percepção de QuorumRESUMO
Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections. Here, by applying native mass spectrometry, we elucidate the effects of lipid substrates on the PG membrane enzymes MraY, MurG, and MurJ. We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C55-P). Further, we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes. Overall, we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention.
Assuntos
Parede Celular , Peptidoglicano , Bactérias , Parede Celular/metabolismo , Lipídeos/química , Peptidoglicano/metabolismoRESUMO
Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.
Assuntos
Ativação do Canal Iônico , Lipídeos de Membrana/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Transporte de Íons , Lipossomos/química , Lipossomos/metabolismo , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Mutação , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Proteína de Transporte de Acila/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure-function studies of metalloid porins, including the basis of their substrate selectivity.
Assuntos
Aquaporinas/química , Proteínas de Arabidopsis/química , Oryza/metabolismo , Raízes de Plantas/metabolismo , Ácido Silícico/metabolismo , Silício/metabolismo , Sequência de Aminoácidos , Aquaporinas/genética , Aquaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Difusão , Expressão Gênica , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Oryza/genética , Raízes de Plantas/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ácido Silícico/química , Silício/química , Especificidade por SubstratoRESUMO
We developed a native mass spectrometry-based approach to quantify the monomer-dimer equilibrium of the LPS transport protein LptH. We use this method to assess the potency and efficacy of an antimicrobial peptide and small molecule disruptors, obtaining new information on their structure-activity relationships. This approach led to the identification of quinoline-based hit compounds representing the basis for the development of novel LPS transport inhibitors.
Assuntos
Anti-Infecciosos/química , Receptores de Lipopolissacarídeos/química , Peptídeos/química , Quinolinas/química , Bibliotecas de Moléculas Pequenas/química , Anti-Infecciosos/farmacologia , Cristalização , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Massas/métodos , Oxazinas/química , Peptídeos/farmacologia , Ligação Proteica , Multimerização Proteica , Quinolinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualization and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. These data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time.
Assuntos
Ligação Competitiva , Proteínas Cromossômicas não Histona/química , DNA Topoisomerase IV/química , Proteínas de Escherichia coli/química , Escherichia coli/químicaRESUMO
The SARS-CoV-2 nucleocapsid (N) protein is a highly immunogenic viral protein that plays essential roles in replication and virion assembly. Here, using native mass spectrometry, we show that dimers are the functional unit of ribonucleoprotein assembly and that N protein binds RNA with a preference for GGG motifs, a common motif in coronavirus packaging signals. Unexpectedly, proteolytic processing of N protein resulted in the formation of additional proteoforms. The N-terminal proteoforms bind RNA, with the same preference for GGG motifs, and bind to cyclophilin A, an interaction which can be abolished by approved immunosuppressant cyclosporin A. Furthermore, N proteoforms showed significantly different interactions with IgM, IgG, and IgA antibodies from convalescent plasma. Notably, the C-terminal proteoform exhibited a heightened interaction with convalescent antibodies, suggesting the antigenic epitope is localized to the C-terminus. Overall, the different interactions of N proteoforms highlight potential avenues for therapeutic intervention and identify a stable and immunogenic proteoform as a possible candidate for immune-directed therapies.
RESUMO
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.
Assuntos
Adesinas Bacterianas/metabolismo , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Adesinas Bacterianas/genética , Aderência Bacteriana , Membrana Celular , Matriz Extracelular , Regulação Bacteriana da Expressão Gênica , Anticorpos de Domínio ÚnicoRESUMO
Membrane proteins are important macromolecules that play crucial roles in many cellular and physiological processes. Over the past two decades, the use of mass spectrometry as a biophysical tool to characterise membrane proteins has grown steadily. By capturing these dynamic complexes in the gas phase, many unknown small molecule interactions have been revealed. One particular application of this research has been the focus on antibiotic resistance with considerable efforts being made to understand underlying mechanisms. Here we review recent advances in the application of mass spectrometry that have yielded both structural and dynamic information on the interactions of antibiotics with proteins involved in bacterial cell envelope biogenesis and drug efflux.
Assuntos
Proteínas de Membrana , Preparações Farmacêuticas , Transporte Biológico , Lipídeos , Espectrometria de Massas , Proteínas de Membrana/metabolismoRESUMO
Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen-deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD ß-taco domain, coupled with conformational changes on ß-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the ß-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the ß-barrel and ß-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipopolissacarídeos/metabolismo , Translocação Genética/genética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Configuração de Carboidratos , Farmacorresistência Bacteriana/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Espectrometria de Massas , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03â µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.
Assuntos
Proteases 3C de Coronavírus/química , Inibidores de Protease de Coronavírus/química , Modelos Moleculares , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica , Sítios de Ligação , Bioensaio , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Cristalografia por Raios X , Espectrometria de Massas , Ligação Proteica , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Replicação ViralRESUMO
The unique architecture of the mycobacterial cell envelope plays an important role in Mycobacterium tuberculosis (Mtb) pathogenesis. A critical protein in cell envelope biogenesis in mycobacteria, required for transport of precursors, trehalose monomycolates (TMMs), is the Mycobacterial membrane protein large 3 (MmpL3). Due to its central role in TMM transport, MmpL3 has been an attractive therapeutic target and a key target for several preclinical agents. In 2019, the first crystal structures of the MmpL3 transporter and its complexes with lipids and inhibitors were reported. These structures revealed several unique structural features of MmpL3 and provided invaluable information on the mechanism of TMM transport. This review aims to highlight the recent advances made in the function of MmpL3 and summarises structural findings. The overall goal is to provide a mechanistic perspective of MmpL3-mediated lipid transport and inhibition, and to highlight the prospects for potential antituberculosis therapies.