Biology subjects

Rubinstein, J. L.

Publications and source records attributed to Rubinstein, J. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of the Arabidopsis thaliana V-type ATPase

Vacuolar-type ATPases (V-ATPases) are evolutionarily conserved rotary proton pumps that play essential roles in the eukaryotic cell. By coupling ATP hydrolysis in their cytosolic V1 region to proton translocation through their membrane-embedded VO region, V-ATPases establish and maintain an acidic pH in the lumen of several different organelles. Functional diversity in the pump is enabled by multiple paralogous genes for the subunits of the complex, which are expressed in a tissue- and organelle-specific manner. Interactions between V-ATPase and TLDc domain-containing proteins have been shown to regulate the enzyme in yeast and mammals but their relevance in plants has remained unclear. We isolated the endogenous V-ATPase from Arabidopsis thaliana leaves and determined its structure by electron cryomicroscopy. Mass spectrometry showed that most of the enzyme originated from the tonoplast. The structural analysis revealed the full rotary catalytic cycle of the plant V-ATPase, and a combination of structural and biochemical experiments showed S-acylation of subunits AP1 and the tonoplast-specific subunit a3 isoform. A subpopulation of complexes derived from the trans-Golgi network/early endosome was identified and found to bind the TLDc protein OXR5. Together, these findings reveal plant-specific features in V-ATPase and suggest organelle-specific interactions with TLDc proteins, pointing to conserved but context-dependent V-ATPase regulation in eukaryotes.

biochemistry↗

bioRxiv2026

The dynamic and heterogeneous structure of the non-canonical inflammasome

Inflammasomes are high molecular weight complexes that play an integral role in the innate immune system, triggering an inflammatory cascade to protect against cellular stresses such as pathogenic bacteria. Both canonical and non-canonical inflammasomes have been described in the literature and detailed structural studies of many components of the more complex and larger canonical versions have been reported. In contrast, corresponding structures of the non-canonical inflammasome have not emerged even though it consists of only two components: lipopolysaccharide (LPS) from gram-negative bacteria, and one of caspase-4 or caspase-5 in humans or caspase-11 in mice. Here we determine the stoichiometry of the non-canonical inflammasome using size-exclusion chromatography coupled with UV, refractive index, and light-scattering measurements, showing that the non-canonical inflammasome is heterogeneous, comprised of three major complexes with different numbers of LPS and caspase molecules. Solution Nuclear Magnetic Resonance (NMR) spectroscopy studies of the N-terminal Caspase Activation and Recruitment Domain (CARD) of caspase-11, that binds LPS, show that it is largely unstructured in the absence of lipid, with pervasive dynamics on the s-ms timescale. Formation of this complex increases the alpha-helical content of the CARD but the dynamics persist, multiple conformers are formed, and tertiary contacts are transient, consistent with formation of a molten globule. Our NMR results establish that the protease domain of caspase-11 is monomeric in isolation. As proteolysis is linked with dimerization, the protease domains are inactive in this state, but upon formation of the non-canonical inflammasome dimerization occurs, priming the complex for rapid processing of substrates. Significance StatementAnimals respond to injury, infection, or toxic materials via a process called inflammation. At the molecular level inflammation involves formation of large machines - inflammasomes - that are instrumental in triggering cascades that lead to an immune response. Although structural studies of canonical inflammasomes have emerged, much less is known about the structures of non-canonical inflammasomes. Using solution Nuclear Magnetic Resonance (NMR) spectroscopy in concert with other biophysical approaches we show that non-canonical inflammasomes are highly dynamic and structurally heterogeneous, and we characterize the different sized inflammasome particles that are formed in terms of their composition. We also show that once formed, non-canonical inflammasomes are primed to rapidly cleave substrate molecules, necessary for propagating the immune response.

biophysics↗

bioRxiv2026

PRRC2A, PRRC2B and PRRC2C are Stress Granule Proteins that Promote Translation Through Association with the eIF3 complex

Regulation of mRNA translation is essential for cellular homeostasis, and its dysregulation contributes to cancer, neurodegeneration, and developmental disorders. Stress granules are cytosolic condensates that form during stress-induced translation arrest and are enriched in mRNAs, translation factors, and RNA-binding proteins, but how stress granule proteins modulate translation remains poorly understood. Here, we identify the stress granule components Proline-Rich Coiled-Coil A, B, and C (PRRC2 proteins) as translation regulators. PRRC2 proteins are large, intrinsically disordered paralogs conserved across jawed vertebrates. Functional proteomics revealed that all PRRC2 proteins associate with the 48S translation initiation complex (PIC), whereas PRRC2B additionally interacts with nuclear proteins. Under stress, the proximal interaction network of PRRC2 proteins undergoes dynamic remodeling, including increased interactions with the stress granule scaffold G3BP1. Genetic perturbation shows that the PRRC2 proteins influence stress granule assembly in a context-specific manner, and are collectively required for cell growth in basal conditions due to their essential role in translation. Cells with reduced PRRC2 proteins exhibit a significant reduction in the abundance of more than half of the proteome, with a bias toward translational targets of eIF3d and eIF4G2. Interaction domain mapping and AlphaFold3 modeling revealed that an helix within the putative coiled-coil domain of PRRC2C mediates interactions with the eIF3 core complex. This modeling places the PRRC2C helix in a previously unassigned region of a published cryo-EM density map, validating the protein interaction and the mechanistic role of PRRC2C in translation control. Together, these findings establish PRRC2 proteins as components of the translation initiation machinery that regulate translation through their interactions with the eIF3 complex and other components of the 48S PIC factors, providing a direct mechanistic link between stress granule proteins and translational control.

cell biology↗

bioRxiv2025

Structural basis for substrate specificity and MSMEG_0435-0436 binding by the mycobacterial long-chain acyl-CoA carboxylase complex

The presence of mycolic acid is a defining feature of the mycobacterial cell wall, which provides a highly impermeable barrier to many antibiotics. Biosynthesis of this fatty acid, as well as tuberculostearic acid, requires precursor molecules produced by the essential long-chain acyl-coenzyme A (CoA) carboxylase (LCC) complex. The LCC complex catalyzes carboxylation of the -carbon of long-chain acyl-CoA, but also short-chain acetyl-CoA and propionyl-CoA. The complex includes the subunits AccA3, which contains a biotin carboxylase (BC) domain and a biotin carboxyl carrier protein (BCCP) domain, the long-chain acyl-CoA carboxyltransferase AccD4, the short-chain acyl-CoA carboxyltransferase AccD5, and the incompletely characterized protein AccE. We used electron cryomicroscopy (cryo-EM) to determine structures of the LCC complex from Mycobacterium smegmatis. In the structures, two AccE subunits tether eight AccA3 subunits to an AccD42AccD54 heterohexamer core. Cryo-EM of the enzyme during catalysis reveals how AccD4 and AccD5 achieve substrate specificity, with AccD5 binding tightly to CoA and AccD4 binding long acyl chains. The BCCP domains of AccA3 undergo long-distance translocation to transfer a carboxyl group from the BC domain of AccA3 to the acyl-CoA substrate bound in AccD5. Further, we find that two copies of a protein complex formed from MSMEG_0435 and MSMEG_0436 can bind the LCC complex, sequestering the biotin moiety of BCCP domains near AccD5. Rv0263c, the Mycobacterium tuberculosis ortholog of MSMEG_0435, has a role in bacterial survival during transmission, suggesting that these proteins may regulate production of branched fatty acid precursors for the mycobacterial cell wall.

biochemistry↗

bioRxiv2025

Mycobacterial EtfD contains an unusual linear cluster and enables β-oxidation to drive proton pumping by the electron transport chain

In mycobacteria, the protein EtfD is thought to link {beta}-oxidation of fatty acids with the electron transport chain, two processes that have attracted attention as targets for therapeutics to treat tuberculosis (TB) and other mycobacterial infections. It has been proposed that targeting {beta}- oxidation could shorten treatment duration by killing non-replicating Mycobacterium tuberculosis within granulomas in the lungs. Here we show that Mycobacterium smegmatis, a fast growing and nonpathogenic model for energy metabolism in M. tuberculosis, relies on EtfD for extracting energy from {beta}-oxidation. Electron cryomicroscopy allowed structure determination of M. smegmatis EtfD, revealing an unusual linear [3Fe-4S] cluster that has not been seen in other protein structures, but which resembles the catalytic noncubane [4Fe-4S] clusters in heterodisulfide reductases. The structure suggests how EtfD transfers electrons from {beta}-oxidation to the electron transport chain. We devised an assay that couples EtfD activity to a fluorescent readout of proton pumping by the electron transport chain, which can be used to identify compounds that block mycobacteria from using {beta}-oxidation to power oxidative phosphorylation.

biochemistry↗

bioRxiv2025

Structure of endogenous Pfs230:Pfs48/45 in complex with potent malaria transmission-blocking antibodies

The Pfs230:Pfs48/45 complex forms the basis for leading malaria transmission-blocking vaccine candidates, yet li]le is known about its molecular assembly. Here, we used cryogenic electron microscopy to elucidate the structure of the endogenous Pfs230:Pfs48/45 complex bound to six potent transmission-blocking antibodies. Pfs230 consists of multiple domain clusters rigidified by interactions mediated through insertion domains. Membrane-anchored Pfs48/45 forms a disc-like structure and interacts with a short C-terminal peptide on Pfs230 that is critical for Pfs230 membrane-retention in vivo. Interestingly, membrane retention through this interaction is not essential for transmission to mosquitoes, suggesting that complex disruption is not a mode of action for transmission-blocking antibodies. Analyses of Pfs48/45-and Pfs230-targeted antibodies identify conserved epitopes on the Pfs230:Pfs48/45 complex and provides a structural paradigm for complement-dependent activity of Pfs230-targeting antibodies. Altogether, the antibody-bound Pfs230:Pfs48/45 structure presented improves our molecular understanding of this biological complex, informing the development of next-generation Plasmodium falciparum transmission-blocking interventions.

molecular biology↗

bioRxiv2025

Structure of mycobacterial NDH-2 bound to a 2-mercapto-quinazolinone inhibitor

The mycobacterial type II NADH dehydrogenase (NDH-2) is a promising drug target because of its central role in energy metabolism in Mycobacterium tuberculosis and other pathogens, and because it lacks a known mammalian homologue. However, the absence of structural information on how the enzyme binds inhibitors has made optimization of lead compounds challenging. We used electron cryomicroscopy (cryo-EM) to determine the structure of NDH-2 from Mycobacterium smegmatis, a fast-growing non-pathogenic model for respiration in M. tuberculosis, both alone and in complex with a 2-mercapto-quinazolinone inhibitor. The structure shows that active mycobacterial NDH-2 is dimeric, with the dimerization interface stabilized by an extended C-terminal helix not found in NDH-2 from other bacterial genera. The arrangement of monomers in the dimer differs from the arrangement described for other prokaryotic NDH-2 dimers, instead resembling dimers formed by NDH-2 in eukaryotes. Density for the 2-mercapto-quinazolinone in the menaquinone-binding site shows that the inhibitor blocks menaquinone reduction through direct interaction with the flavin adenine dinucleotide cofactor. These results reveal structural elements of NDH-2 that could be used to design specific inhibitors of the mycobacterial enzyme.

biochemistry↗

bioRxiv2024

Coordinated conformational changes in the Tad pilus ATPase CpaF facilitate a rotary mechanism of catalysis

The type IV pilus family uses PilT-like ATPases to rapidly assemble and disassemble pilin subunits. Among them, the tight adherence (Tad) pilus performs both functions using a single bifunctional ATPase, CpaF. How ATP catalysis by CpaF facilitates both assembly and disassembly of the Tad pilus remains unclear. Here, we determined electron cryo-microscopy structures of CpaF in three distinct conformations and nucleotide occupancies. Packing unit analyses revealed differences in nucleotide coordination in the active sites, as well as synchronized domain movements during the catalytic cycle. Alphafold3 modeling demonstrated that CpaF and other motors within the type IV filament superfamily use a similar binding interface to engage their respective platform proteins. From these data, we propose that CpaF employs a clockwise, rotary mechanism of catalysis, which couples chemical energy to mechanical force during the biogenesis of Tad pili, a process broadly applicable to other single motor systems.

microbiology↗

bioRxiv2024

Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria

Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis and enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III2IV2 (CIII2CIV2) supercomplex. Analysis of the Mqo:CIII2CIV2 interaction shows that CIII2CIV2 is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII2CIV2 enables electron transfer from malate to CIII2CIV2 with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.

biochemistry↗

bioRxiv2024

De novo design of potent inhibitors of Clostridioides difficile toxin B

Clostridioides difficile is a major cause of secondary disease in hospitals. During infection, C. difficile toxin B drives disease pathology. Here we use deep learning and Rosetta-based approaches to de novo design small proteins that block the entry of TcdB into cells. These molecules have binding affinities and neutralization IC50s in the pM range and are compelling candidates for further clinical development. By directly targeting the toxin rather than the pathogen, these molecules have the advantage of immediate cessation of disease and lower selective pressure for escape compared to conventional antibiotics. As C. difficile infects the colon, the protease and pH resistance of the designed proteins opens the door to oral delivery of engineered biologics. Significance statementC. difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in 30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection (rCDI). Progress has been made in the development of anti-toxin mAb therapies that can reduce the rate of rCDI, but these remain unaffordable and out of reach for many patients. Using de novo protein design, we developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. These molecules are high affinity, potently neutralizing and stable in simulated intestinal fluid, making them strong candidates for the clinical development of new CDI therapies.

microbiology↗

bioRxiv2024

Structure of yeast RAVE bound to a partial V1 complex

Vacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multi-subunit enzymes consist of a soluble catalytic V1 subcomplex and a membrane-embedded proton-translocating VO subcomplex. The V1 and VO subcomplexes can undergo reversible dissociation to regulate proton pumping, with reassociation of V1 and VO requiring the protein complex known as RAVE (regulator of the ATPase of vacuoles and endosomes). In the yeast Saccharomyces cerevisiae, RAVE consists of subunits Rav1p, Rav2p, and Skp1p. We used electron cryomicroscopy (cryo-EM) to determine a structure of yeast RAVE bound to V1. In the structure, RAVE is a L-shaped complex with Rav2p pointing toward the membrane and Skp1p distant from both the membrane and V1. Only Rav1p interacts with V1, binding to a region of subunit A not found in the corresponding ATP synthase subunit. When bound to RAVE, V1 is in a rotational state suitable for binding the free VO complex, but it is partially disrupted in the structure, missing five of its 16 subunits. Other than these missing subunits and the conformation of the inhibitory subunit H, the V1 complex with RAVE appears poised for reassembly with VO.

biochemistry↗

bioRxiv2024

Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles

Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures of membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs of these vesicles can be challenging due to the strong signal contributed from the lipid bilayer. This challenge often requires iterative and laborious particle selection workflows to generate a dataset of high-quality particle images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on the Segment Anything model. Vesicle Picker enables automatic identification of vesicles in cryo-EM micrographs with high recall and precision. It then exhaustively selects all potential particle locations, either at the perimeter or uniformly over the surface of the projection of the vesicle. The program is designed to interface with cryoSPARC, which performs both upstream micrograph processing and downstream single particle image analysis. We demonstrate Vesicle Pickers utility by determining a high-resolution map of the vacuolar-type ATPase from micrographs of native synaptic vesicles (SVs) and identifying an additional protein or protein complex in the SV membrane.

biophysics↗

bioRxiv2024

High resolution cryo-EM of V-ATPase in native synaptic vesicles

Neurotransmitter release into the synaptic cleft between neurons enables the intercellular communication central to the function of the brain. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single particle electron cryomicroscopy of the vesicles allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzymes rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. Conditions where ATP hydrolysis drives glutamate loading result in loss of V1 from the SV membrane, suggesting that SV loading is sufficient to induce V-ATPase dissociation.

biochemistry↗

bioRxiv2023

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically de-acidified compartments targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to pH gradient disruption is unclear. Here, we reveal a direct role for the V1H subunit of the V-ATPase proton pump in recruiting ATG16L1. The interaction between V1H and ATG16L1 occurs within assembled V-ATPases, but not dissociated V1 complexes. This selectivity allows recruitment to be coupled to changes in V-ATPase assembly that follow pH dissipation. Cells lacking V1H undergo canonical macroautophagy but are unable to recruit ATG16L1 in response to influenza infection or ionophore drugs. We identify a loop within V1H that mediates ATG16L1 binding, which is absent in a neuronal isoform of V1H. Thus, V1H controls ATG16L1 recruitment in response to proton gradient dissipation, suggesting that the V-ATPase acts autonomously as a cell-intrinsic damage sensor.

molecular biology↗

bioRxiv2023

Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin- RING E3 ligase complex (CRL3KCTD5) that ubiquitylates G{beta}{gamma} and reduces G{beta}{gamma} protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/G{beta}1{gamma}2 assembly reveals a highly dynamic complex with rotations of over 60{degrees} between the KCTD5BTB/CUL3NTD and KCTD5CTD/G{beta}{gamma} moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate G{beta}{gamma} in an E3-E3 super-assembly, and extension of the structure to include full- length CUL3 with RBX1 and an ARIH1[~]ubiquitin conjugate reveals that some conformational states position the ARIH1[~]ubiquitin thioester bond to within 10 [A] of lysine-23 of G{beta} and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/G{beta}{gamma} complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex. Significance StatementIn humans, [~]600 enzyme complexes can carry out protein ubiquitylation, and the most abundant class of these are the cullin3-RING-ligase complexes (CRL3s). CRL3s are multiprotein complexes built around a BTB/cullin3 core, and the incorporation of different BTB proteins into this scaffold results in distinct architectures that ubiquitylate a wide range of substrates. In most cases, it is not known how the complexes are tuned to their substrates. We show that the BTB protein KCTD5 is the central organizer in a CRL3KCTD5 complex, and that the architecture and internal dynamics of KCTD5 are essential for positioning a G{beta}{gamma} substrate protein near an activated ubiquitin for the transfer reaction. This explains how KCTD5 targets G{beta}{gamma} for proteasomal degradation and regulates cellular activities.

biochemistry↗

bioRxiv2023

A simple assay for inhibitors of mycobacterial oxidative phosphorylation

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and adenosine triphosphate (ATP) synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and non-specific membrane uncoupling. In this assay, IMVs from M. smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Non-specific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2 while thioridazine uncouples the mycobacterial membrane non-specifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

biochemistry↗

bioRxiv2023

Structure of a dimeric ABC transporter

ATP binding cassette (ABC) proteins generally couple ATP hydrolysis to the active transport of solutes across cellular membranes. All ABC proteins contain a core structure of two transmembrane domains (TMD1, TMD2) and two nucleotide binding domains (NBD1, NBD2), and many ABC proteins contain additional domains. Some members of the C subfamily of ABC (ABCC) proteins, such as the multidrug resistant protein 1 (MRP1), contain an N-terminal transmembrane domain (TMD0) and L0 linker that regulate transport activity and cellular trafficking, and mediate interactions with other proteins. Regulation can also be imparted by phosphorylation, proteolytic processing, and/or oligomerization of the proteins. Here we present the structure of yeast cadmium factor 1 (Ycf1p), a homologue of MRP1, in its mature form following cleavage by the yeast protease Pep4p. Remarkably, proteolytically cleaved Ycf1p forms a well-ordered dimer, with some monomeric particles also present in solution. Numerous other ABC proteins have been proposed to form dimers but no high-resolution structures have been reported. The monomeric and dimeric Ycf1p species are differentially phosphorylated at the intrinsically disordered regulatory (R) region, which links NBD1 to TMD2, and possess different ATPase activities indicating that dimerization affects the function of the protein. Protein-protein interactions involving TMD0, the L0 linker, and the R region mediate contacts between Ycf1p protomers in the dimer. In addition, cryo-EM density is observed for lipids at the interface between protomers, which suggests that lipids stabilize the dimer. The Ycf1p dimer structure is consistent with proposed dimerization interfaces of other ABCC dimers, such as MRP1.

biochemistry↗

bioRxiv2023

Structural basis for inhibition of mycobacterial ATP synthase by squaramides and second generation diarylquinolines

Mycobacteria, such as Mycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction of Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared to BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzymes proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines have been reported to kill mycobacteria while SQ31f has not.

biochemistry↗