Biology subjects

Rubinstein, J. L.

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

50 records · Page 3Linked to original sources

bioRxiv2020

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

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 that maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred between respiratory complexes III and IV (CIII and CIV) by water-soluble cyt. c. In S. cerevisiae and some other organisms, these complexes assemble into larger CIII2CIV1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplexs cyt.c-mediated QH2:O2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Cryo-EM structures of the supercomplex with cyt. c reveal distinct states where the positively-charged cyt. c is bound either to CIII or CIV, or resides at intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively-charged surface patch of the supercomplex. Thus, rather than enhancing electron-transfer rates by decreasing the distance cyt. c must diffuse in 3D, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by 2D diffusion of cyt. c. This mechanism enables the CIII2CIV1/2 supercomplex to increase QH2:O2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain. Significance StatementIn the last steps of food oxidation in living organisms, electrons are transferred to oxygen through the membrane-bound respiratory chain. This electron transfer is mediated by mobile carriers such as membrane-bound quinone and water-soluble cyt. c. The latter transfers electrons from respiratory complex III to IV. In yeast these complexes assemble into III2IV1/2 supercomplexes, but their role has remained enigmatic. This study establishes a functional role for this supramolecular assembly in the mitochondrial membrane. We used cryo-EM and kinetic studies to show that cyt. c shuttles electrons by sliding along the surface of III2IV1/2 (2D diffusion). The structural arrangement into III2IV1/2 supercomplexes suggests a mechanism to regulate cellular respiration.

biochemistry↗

bioRxiv2020

Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus Disease 2019 (COVID-19), has caused a global pandemic. Antibodies are powerful biotherapeutics to fight viral infections; however, discovery of the most potent and broadly acting clones can be lengthy. Here, we used 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 (IC50) values as low as 9 x 10-14 M were achieved as a result of up to 10,000-fold potency enhancements. 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 Ig-like in vivo bioavailability. This MULTi-specific, multi-Affinity antiBODY (Multabody; or MB) platform contributes a new class of medical countermeasures against COVID-19 and an efficient approach to rapidly deploy potent and broadly-acting therapeutics against infectious diseases of global health importance. One Sentence Summarymultimerization platform transforms antibodies emerging from discovery screens into potent neutralizers that can overcome SARS-CoV-2 sequence diversity.

molecular biology↗

bioRxiv2020

Structure of mycobacterial ATP synthase with the TB drug bedaquiline

Tuberculosis (TB), the leading cause of death by infectious disease worldwide, is increasingly resistant to first line antibiotics. Developed from a screen against Mycobacterium smegmatis, bedaquiline can sterilize even latent M. tuberculosis infections that may otherwise persist for decades and has become a cornerstone of treatment for multidrug resistant and extensively-drug resistant TB. Bedaquiline targets mycobacterial ATP synthase, an essential enzyme in the obligate aerobic Mycobacterium genus. However, how the drug binds the intact enzyme is unknown. We determined the structure of M. smegmatis ATP synthase with and without bedaquiline. The drug-free structure reveals hook-like extensions from the enzymes subunits that inhibit ATP hydrolysis in low-energy conditions, such as during latent infections. Bedaquiline binding induces global conformational changes in ATP synthase, creating tight binding pockets at the interface of subunits a and c. These binding sites explain the drugs structure-activity relationship and its potency as an antibiotic for TB.

biochemistry↗

bioRxiv2020

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

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 P. falciparum (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 P. berghei 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.

microbiology↗

bioRxiv2020

TsaP binds PilQ and is involved in cyclic di-GMP signaling

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fibre 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 fibre 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 [A] and 4.4 [A] resolution in the presence and absence of C7 symmetric spokes, respectively. The heptameric spokes were found to be the two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while the protein is not essential for twitching motility, over-expression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spokes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade. IMPACT STATEMENTThe type IV pilus is critical for bacterial virulence. The co-structure of the pilus secretin PilQ and TsaP is determined. Characterization of TsaP implicates it in surface-sensing signal transduction.

microbiology↗

bioRxiv2020

Through-grid wicking enables high-speed cryoEM specimen preparation

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. We show 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. We combined ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device that we name Back-it-up, or BIU. The high liquid-absorption capacity of the glass fiber compared to self-wicking grids appears to make the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The devices speed increases the number of views for a specimen that suffers from preferred orientations.

biophysics↗

bioRxiv2020

Electron Event Representation (EER) data enables efficient cryoEM file storage with full preservation of spatial and temporal resolution

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/sec) to camera exposure rate (electrons/pixel/sec) allows electron counting, which further improves DQE and enables recording of super-resolution information. Movie output also allows for computational 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 file size, to one suitable for storage and image processing. This reduction in the cameras temporal resolution 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 we present experimental analysis of a new Electron Event Representation (EER) data format for electron counting DDD movies, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables 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 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.

biophysics↗

bioRxiv2019

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

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis 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 the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel 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 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. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key {beta}-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 upon 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 inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis. Significance StatementThe MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.

biophysics↗

bioRxiv2019

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

Clostridium sordellii lethal toxin (TcsL) is responsible for an almost invariably lethal toxic shock syndrome associated with gynecological C. sordellii infections. Here, using CRISPR/Cas9 screening, we identify semaphorins SEMA6A and SEMA6B as the cellular receptors for TcsL and demonstrate that soluble extracellular SEMA6A can protect mice from TcsL-induced edema. A 3.3 [A] cryo-EM structure shows that TcsL binds SEMA6A with the same region that the highly related C. difficile TcdB toxin uses to bind structurally unrelated Frizzled receptors. Remarkably, reciprocal mutations in this evolutionarily divergent surface are sufficient to switch receptor specificity between the toxins. 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.

molecular biology↗

bioRxiv2019

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

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 Neisseria meningitidis at 2.3 to 3.3 [A] resolution. The structures allow development of a model in which the cyclical hydrolysis of ATP is coupled to concerted 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.

biophysics↗

bioRxiv2019

Multiple conformations facilitate PilT function in the type IV pilus

Type IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.

microbiology↗

bioRxiv2019

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

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 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 we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. 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 straightforward customization of the instrument for specialized experiments.\n\nSynopsisA method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

biophysics↗

bioRxiv2018

Structure of a bacterial ATP synthase

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 Bacillus PS3 ATP synthase in Eschericia coli, 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{varepsilon} 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.

biochemistry↗

bioRxiv2017

Structure of a AAA+ unfoldase in the process of unfolding substrate

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 [A] stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

biochemistry↗