Publications by authors named "John L Rubinstein"

89 Publications

Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers.

Nat Commun 2021 06 16;12(1):3661. Epub 2021 Jun 16.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC) values as low as 9 × 10 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-23825-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209050PMC
June 2021

Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.

Biochemistry 2021 Jun 3;60(23):1808-1821. Epub 2021 Jun 3.

Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (F) specific for the TSC2 subunit of pTSC. We demonstrate F-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 μm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.
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http://dx.doi.org/10.1021/acs.biochem.1c00269DOI Listing
June 2021

Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.

Proc Natl Acad Sci U S A 2021 May;118(21)

Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada;

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.
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http://dx.doi.org/10.1073/pnas.2025853118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166025PMC
May 2021

Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome in the yeast III-IV respiratory supercomplex.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden;

Energy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport, which maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred from respiratory complexes III to IV (CIII and CIV) by water-soluble cytochrome (cyt.) In and some other organisms, these complexes assemble into larger CIIICIV supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the supercomplex cyt. -mediated QH:O oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. show the positively charged cyt. bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. must diffuse in three dimensions, formation of the CIIICIV supercomplex facilitates electron transfer by two-dimensional (2D) diffusion of cyt. This mechanism enables the CIIICIV supercomplex to increase QH:O oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.
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http://dx.doi.org/10.1073/pnas.2021157118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980474PMC
March 2021

CryoEM map of Pseudomonas aeruginosa PilQ enables structural characterization of TsaP.

Structure 2021 05 17;29(5):457-466.e4. Epub 2020 Dec 17.

Program in Molecular Structure & Function, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada. Electronic address:

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fiber used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fiber out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 Å and 4.4 Å resolution in the presence and absence of C symmetric spikes, respectively. The heptameric spikes were found to be two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while it is not essential for twitching motility, overexpression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spikes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade.
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http://dx.doi.org/10.1016/j.str.2020.11.019DOI Listing
May 2021

Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline.

Nature 2021 01 9;589(7840):143-147. Epub 2020 Dec 9.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada.

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis. This drug can sterilize even latent M. tuberculosis infections and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Bedaquiline targets the mycobacterial ATP synthase, which is an essential enzyme in the obligate aerobic Mycobacterium genus, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.
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http://dx.doi.org/10.1038/s41586-020-3004-3DOI Listing
January 2021

Revised subunit order of mammalian septin complexes explains their in vitro polymerization properties.

Mol Biol Cell 2021 02 2;32(3):289-300. Epub 2020 Dec 2.

Cell Biology Program, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.

Septins are conserved GTP-binding cytoskeletal proteins that polymerize into filaments by end-to-end joining of hetero-oligomeric complexes. In human cells, both hexamers and octamers exist, and crystallography studies predicted the order of the hexamers to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7, while octamers are thought to have the same core, but with SEPT9 at the ends. However, based on this septin organization, octamers and hexamers would not be expected to copolymerize due to incompatible ends. Here we isolated hexamers and octamers of specific composition from human cells and show that hexamers and octamers polymerize individually and, surprisingly, with each other. Binding of the Borg homology domain 3 (BD3) domain of Borg3 results in distinctive clustering of each filament type. Moreover, we show that the organization of hexameric and octameric complexes is inverted compared with its original prediction. This revised septin organization is congruent with the organization and behavior of yeast septins suggesting that their properties are more conserved than was previously thought.
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http://dx.doi.org/10.1091/mbc.E20-06-0398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8098831PMC
February 2021

Structural ordering of the circumsporozoite protein repeats by inhibitory antibody 3D11.

Elife 2020 11 30;9. Epub 2020 Nov 30.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, Canada.

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 - the leading malaria vaccine against (Pf) - has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.
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http://dx.doi.org/10.7554/eLife.59018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704109PMC
November 2020

A pH-Dependent Conformational Switch Controls ClpP Protease Function.

J Am Chem Soc 2020 12 24;142(49):20519-20523. Epub 2020 Nov 24.

Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure-function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen . Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.
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http://dx.doi.org/10.1021/jacs.0c09474DOI Listing
December 2020

Through-grid wicking enables high-speed cryoEM specimen preparation.

Acta Crystallogr D Struct Biol 2020 Nov 13;76(Pt 11):1092-1103. Epub 2020 Oct 13.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada.

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
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http://dx.doi.org/10.1107/S2059798320012474DOI Listing
November 2020

An intrinsically disordered motif regulates the interaction between the p47 adaptor and the p97 AAA+ ATPase.

Proc Natl Acad Sci U S A 2020 10 7;117(42):26226-26236. Epub 2020 Oct 7.

Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada;

VCP/p97, an enzyme critical to proteostasis, is regulated through interactions with protein adaptors targeting it to specific cellular tasks. One such adaptor, p47, forms a complex with p97 to direct lipid membrane remodeling. Here, we use NMR and other biophysical methods to study the structural dynamics of p47 and p47-p97 complexes. Disordered regions in p47 are shown to be critical in directing intra-p47 and p47-p97 interactions via a pair of previously unidentified linear motifs. One of these, an SHP domain, regulates p47 binding to p97 in a manner that depends on the nucleotide state of p97. NMR and electron cryomicroscopy data have been used as restraints in molecular dynamics trajectories to develop structural ensembles for p47-p97 complexes in adenosine diphosphate (ADP)- and adenosine triphosphate (ATP)-bound conformations, highlighting differences in interactions in the two states. Our study establishes the importance of intrinsically disordered regions in p47 for the formation of functional p47-p97 complexes.
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http://dx.doi.org/10.1073/pnas.2013920117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585011PMC
October 2020

Electron-event representation data enable efficient cryoEM file storage with full preservation of spatial and temporal resolution.

IUCrJ 2020 Sep 7;7(Pt 5):860-869. Epub 2020 Aug 7.

Molecular Medicine Program, The Hospital for Sick Children, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.

Direct detector device (DDD) cameras have revolutionized electron cryomicroscopy (cryoEM) with their high detective quantum efficiency (DQE) and output of movie data. A high ratio of camera frame rate (frames per second) to camera exposure rate (electrons per pixel per second) allows electron counting, which further improves the DQE and enables the recording of super-resolution information. Movie output also allows the correction of specimen movement and compensation for radiation damage. However, these movies come at the cost of producing large volumes of data. It is common practice to sum groups of successive camera frames to reduce the final frame rate, and therefore the file size, to one suitable for storage and image processing. This reduction in the temporal resolution of the camera requires decisions to be made during data acquisition that may result in the loss of information that could have been advantageous during image analysis. Here, experimental analysis of a new electron-event representation (EER) data format for electron-counting DDD movies is presented, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables the recording of DDD movies at the raw camera frame rate without sacrificing either spatial or temporal resolution. Experimental data demonstrate that the method retains super-resolution information and allows the correction of specimen movement at the physical frame rate of the camera while maintaining manageable file sizes. The EER format will enable the development of new methods that can utilize the full spatial and temporal resolution of DDD cameras.
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http://dx.doi.org/10.1107/S205225252000929XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467176PMC
September 2020

Probing Cooperativity of N-Terminal Domain Orientations in the p97 Molecular Machine: Synergy Between NMR Spectroscopy and Cryo-EM.

Angew Chem Int Ed Engl 2020 12 6;59(50):22423-22426. Epub 2020 Oct 6.

Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.

The hexameric p97 enzyme plays an integral role in cellular homeostasis. Large changes to the orientation of its N-terminal domains (NTDs), corresponding to NTD-down (p97-ADP) or NTD-up (p97-ATP), accompany ATP hydrolysis. The NTDs in a series of p97 disease mutants interconvert rapidly between up and down conformations when p97 is in the ADP-bound state. While the populations of up and down NTDs can be determined from bulk measurements, information about the cooperativity of the transition between conformations is lacking. Here we use cryo-EM to determine populations of the 14 unique up/down NTD states of the homo-hexameric R95G disease-causing p97 ring, showing that NTD orientations do not depend on those of neighboring subunits. In contrast, NMR studies establish that inter-protomer cooperativity is important for regulating the orientation of NTDs in p97 particles comprising mixtures of different subunits, such as wild-type and R95G, emphasizing the synergy between cryo-EM and NMR in establishing how the components of p97 function.
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http://dx.doi.org/10.1002/anie.202009767DOI Listing
December 2020

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell 2020 07 25;182(2):345-356.e16. Epub 2020 Jun 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Molecular Architecture of Life Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada. Electronic address:

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
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http://dx.doi.org/10.1016/j.cell.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316060PMC
July 2020

Structure of V-ATPase from the mammalian brain.

Science 2020 03;367(6483):1240-1246

Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada.

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a effector protein. Cryo-electron microscopy allowed the construction of an atomic model, defining the enzyme's ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions.
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http://dx.doi.org/10.1126/science.aaz2924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324285PMC
March 2020

An allosteric switch regulates ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.

Proc Natl Acad Sci U S A 2020 03 2;117(11):5895-5906. Epub 2020 Mar 2.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;

The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target .
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http://dx.doi.org/10.1073/pnas.1921630117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084164PMC
March 2020

Structure and Roles of V-type ATPases.

Trends Biochem Sci 2020 04 28;45(4):295-307. Epub 2020 Jan 28.

The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Biochemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Medical Biophysics, The University of Toronto, Toronto, ON M5G 1L7, Canada. Electronic address:

V-ATPases are membrane-embedded protein complexes that function as ATP hydrolysis-driven proton pumps. V-ATPases are the primary source of organellar acidification in all eukaryotes, making them essential for many fundamental cellular processes. Enzymatic activity can be modulated by regulated and reversible disassembly of the complex, and several subunits of mammalian V-ATPase have multiple isoforms that are differentially localized. Although the biochemical properties of the different isoforms are currently unknown, mutations in specific subunit isoforms have been associated with various diseases, making V-ATPases potential drug targets. V-ATPase structure and activity have been best characterized in Saccharomyces cerevisiae, where recent structures have revealed details about the dynamics of the enzyme, the proton translocation pathway, and conformational changes associated with regulated disassembly and autoinhibition.
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http://dx.doi.org/10.1016/j.tibs.2019.12.007DOI Listing
April 2020

A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery.

Elife 2020 01 9;9. Epub 2020 Jan 9.

Department of Biochemistry, University of Toronto, Toronto, Canada.

The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.
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http://dx.doi.org/10.7554/eLife.52158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112952PMC
January 2020

Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

Acta Crystallogr D Struct Biol 2019 Dec 22;75(Pt 12):1063-1070. Epub 2019 Nov 22.

The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.
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http://dx.doi.org/10.1107/S2059798319014372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889916PMC
December 2019

Multiple conformations facilitate PilT function in the type IV pilus.

Nat Commun 2019 11 15;10(1):5198. Epub 2019 Nov 15.

Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.

Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C symmetry; however, most of these ATPases crystallize with either C or C symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C C, and C conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.
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http://dx.doi.org/10.1038/s41467-019-13070-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858323PMC
November 2019

Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response.

Sci Transl Med 2019 10;11(516)

Princess Margaret Cancer Centre, Toronto, Ontario M5G 1L7, Canada.

Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.
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http://dx.doi.org/10.1126/scitranslmed.aax2863DOI Listing
October 2019

The human coronavirus HCoV-229E S-protein structure and receptor binding.

Elife 2019 10 25;8. Epub 2019 Oct 25.

Department of Molecular Genetics, The University of Toronto, Toronto, Canada.

The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the cross-species transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.
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http://dx.doi.org/10.7554/eLife.51230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970540PMC
October 2019

Structural comparison of the vacuolar and Golgi V-ATPases from .

Proc Natl Acad Sci U S A 2019 04 25;116(15):7272-7277. Epub 2019 Mar 25.

Molecular Medicine Program, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;

Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast , isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that V complexes containing Stv1p could be readily purified without attached V regions. We used this effect to determine structures of the membrane-embedded V region with Stv1p at 3.1-Å resolution, which we compare with a structure of the V region with Vph1p that we determine to 3.2-Å resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.
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http://dx.doi.org/10.1073/pnas.1814818116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462096PMC
April 2019

Structure of a bacterial ATP synthase.

Elife 2019 02 6;8. Epub 2019 Feb 6.

The Hospital for Sick Children Research Institute, Toronto, Canada.

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the PS3 ATP synthase in , purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.
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http://dx.doi.org/10.7554/eLife.43128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377231PMC
February 2019

Cooperative subunit dynamics modulate p97 function.

Proc Natl Acad Sci U S A 2019 01 24;116(1):158-167. Epub 2018 Dec 24.

Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada;

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.
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http://dx.doi.org/10.1073/pnas.1815495116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320498PMC
January 2019

Structure of a functional obligate complex IIIIV respiratory supercomplex from Mycobacterium smegmatis.

Nat Struct Mol Biol 2018 12 5;25(12):1128-1136. Epub 2018 Dec 5.

Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate IIIIV supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Q and Q sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.
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http://dx.doi.org/10.1038/s41594-018-0160-3DOI Listing
December 2018

Cryo-EM of ATP synthases.

Curr Opin Struct Biol 2018 10 18;52:71-79. Epub 2018 Sep 18.

The Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, M5G 0A4, Canada; Department of Medical Biophysics, The University of Toronto, M5G 1L7, Canada; Department of Biochemistry, The University of Toronto, M5S 1A8, Canada. Electronic address:

ATP synthases are rotary enzymes found in bacteria, chloroplasts, and mitochondria. These complexes produce the majority of cellular ATP in aerobic cells using energy from the transmembrane proton motive force established by the electron transport chain. In mitochondria, dimeric ATP synthase is essential for formation of the inner membrane cristae. While rotary catalysis in the soluble F1 region has been studied extensively by X-ray crystallography, the structure of the membrane embedded FO region remained elusive until recently. In the past few years, electron cryomicroscopy structures of mitochondrial, chloroplast, and bacterial ATP synthases have revealed the architecture of the FO region, helping to explain the mechanisms of proton translocation, dimerization of the enzyme in mitochondria, and cristae formation. These structures also show that ATP synthases exist in different conformational states, illustrating the flexibility and dynamics of the complex.
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http://dx.doi.org/10.1016/j.sbi.2018.08.005DOI Listing
October 2018

Reversible inhibition of the ClpP protease via an N-terminal conformational switch.

Proc Natl Acad Sci U S A 2018 07 25;115(28):E6447-E6456. Epub 2018 Jun 25.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of and ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.
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http://dx.doi.org/10.1073/pnas.1805125115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048506PMC
July 2018

Structure of the alternative complex III in a supercomplex with cytochrome oxidase.

Nature 2018 05 25;557(7703):123-126. Epub 2018 Apr 25.

Department of Biochemistry, University of Illinois, Urbana, IL, USA.

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria. Like complex III (also known as the bc complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily , including the proton pump polysulfide reductase. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer, both as an independent enzyme and as a functional 1:1 supercomplex with an aa-type cytochrome c oxidase (cyt aa). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.
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http://dx.doi.org/10.1038/s41586-018-0061-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004266PMC
May 2018

Band 3 function and dysfunction in a structural context.

Curr Opin Hematol 2018 05;25(3):163-170

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

Purpose Of Review: Current research on the human band 3 glycoprotein, the red cell chloride/bicarbonate anion exchanger (AE1), is highlighted and placed within a structural context.

Recent Findings: The determination of the crystal structure of the membrane domain of human band 3, the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters, is a major breakthrough toward understanding the mechanism of action of this membrane transport protein, its interaction with partner proteins, and how mutations linked to disease affect its ability to fold and function.

Summary: Band 3 contains 14 transmembrane segments arranged in a 7+7 transmembrane inverted repeat topology common to all members of the SLC4 family and the unrelated SLC26 anion transporter family. A functional feature of this fold is the presence of a core and a gate domain: the core domain contains two short transmembrane helices (TM3 and 10) that face each other in the middle of the membrane with the positive N-terminal helix dipoles creating the anion-binding site, whereas the gate domain forms the dimer interface. During transport, the movement of these two domains relative to each other provides the intracellular and extracellular compartments with alternating access to the central anion-binding site.
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http://dx.doi.org/10.1097/MOH.0000000000000418DOI Listing
May 2018
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