Structure of the bc1-cbb3 respiratory supercomplex from Pseudomonas aeruginosa.

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc1) and one of the organism's terminal oxidases, cytochrome cbb3, which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c4 and a single heme cytochrome c5. Together, these proteins allow electron transfer from ubiquinol in cytochrome bc1 to oxygen in cytochrome cbb3. We also present evidence that different isoforms of cytochrome cbb3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc1-cbb3 SCs also exist in other bacterial pathogens.

Flexible Client-Dependent Cages in the Assembly Landscape of the Periplasmic Protease-Chaperone DegP.

The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of density maps and structural models that include those for novel particles of approximately 30 and 60 monomers. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.

Imidazopyridine Amides: Synthesis, Mycobacterium smegmatis CIII2CIV2 Supercomplex Binding, and In Vitro Antimycobacterial Activity.

Q203 (telacebec) is an imidazopyridine amide (IPA) targeting the respiratory CIII2CIV2 supercomplex of the mycobacterial electron transport chain (ETC). Aiming for a better understanding of the molecular mechanism of action of IPA, 27 analogues were prepared through a seven-step synthetic scheme. Oxygen consumption assay was designed to test the inhibition of purified Mycobacterium smegmatis CIII2CIV2 by these compounds. The assay results generally supported structure-activity relationship information obtained from the structure of M. smegmatis CIII2CIV2 bound to Q203. The IC50 of Q203 and compound 27 was 99 ± 32 and 441 ± 138 nM, respectively. All IPAs including Q203 showed no inhibition of mitochondrial ETC, proving their selectivity against mycobacteria. In vitro Mycobacterium tuberculosis growth inhibition and M. smegmatis CIII2CIV2 binding did not correlate perfectly. These observations suggest that further investigation into the mechanisms of resistance in different mycobacterial species is needed to understand the lack of the correlation pattern between CIII2CIV2 inhibition and cellular activity.

Structural basis of mammalian complex IV inhibition by steroids.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme's catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III.

Electron transfer between respiratory complexes drives transmembrane proton translocation, which powers ATP synthesis and membrane transport. The homodimeric respiratory complex III (CIII2) oxidizes ubiquinol to ubiquinone, transferring electrons to cytochrome c and translocating protons through a mechanism known as the Q cycle. The Q cycle involves ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We determined structures of Candida albicans CIII2 by cryoelectron microscopy (cryo-EM), revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions.

Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203).

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.

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, 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.) c In Saccharomyces 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 supercomplex 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. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. c show the positively charged cyt. c bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. c must diffuse in three dimensions, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by two-dimensional (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.

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 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.

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

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.

Integrated Synthetic, Biophysical, and Computational Investigations of Covalent Inhibitors of Prolyl Oligopeptidase and Fibroblast Activation Protein α.

Over the past decade, there has been increasing interest in covalent inhibition as a drug design strategy. Our own interest in the development of prolyl oligopeptidase (POP) and fibroblast activation protein α (FAP) covalent inhibitors has led us to question whether these two serine proteases were equal in terms of their reactivity toward electrophilic warheads. To streamline such investigations, we exploited both computational and experimental methods to investigate the influence of different reactive groups on both potency and binding kinetics using both our own series of POP inhibitors and others' discovered hits. A direct correlation between inhibitor reactivity and residence time was demonstrated through quantum mechanics methods and further supported by experimental studies. This computational method was also successfully applied to FAP, as an overview of known FAP inhibitors confirmed our computational predictions that more reactive warheads (e.g., boronic acids) must be employed to inhibit FAP than for POP.

Inhibition and Activation of Kinases by Reaction Products: A Reporter-Free Assay.

Kinases are widely distributed in nature and are implicated in many human diseases. Thus, an understanding of their activity and regulation is of fundamental importance. Several kinases are known to be inhibited by ADP. However, thorough investigation of this phenomenon is hampered by the lack of a simple and effective assay for studying this inhibition. We now present a quick, general approach for measuring the effects of reaction products on kinase activity. The method, based on isothermal titration calorimetry, is the first universal, reporter-free, continuous assay for probing kinase inhibition or activation by ADP. In applications to an aminoglycoside phosphotransferase [APH(3')-IIIa] and pantothenate kinases from Escherichia coli (EcPanK) and Pseudomonas aeruginosa (PaPanK), we found ADP to be an efficient inhibitor of all three kinases, with inhibition constant (Ki) values similar to or lower than the Michaelis-Menten constant (Km) values of ATP. Interestingly, ADP was an activator at low concentrations and an inhibitor at high concentrations for EcPanK. This unusual effect was quantitatively modeled and attributed to cooperative interactions between the two subunits of the dimeric enzyme. Importantly, our results suggest that, at typical bacterial intracellular concentrations of ATP and ADP (approximately 1.5 mM and 180 µM, respectively), all three kinases are partially inhibited by ADP, allowing enzyme activity to rapidly respond to changes in the levels of both metabolites.

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment.

Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.

Exploring the role of post-translational modifications in regulating α-synuclein interactions by studying the effects of phosphorylation on nanobody binding.

Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.

Rapid measurement of inhibitor binding kinetics by isothermal titration calorimetry.

Although drug development typically focuses on binding thermodynamics, recent studies suggest that kinetic properties can strongly impact a drug candidate's efficacy. Robust techniques for measuring inhibitor association and dissociation rates are therefore essential. To address this need, we have developed a pair of complementary isothermal titration calorimetry (ITC) techniques for measuring the kinetics of enzyme inhibition. The advantages of ITC over standard techniques include speed, generality, and versatility; ITC also measures the rate of catalysis directly, making it ideal for quantifying rapid, inhibitor-dependent changes in enzyme activity. We used our methods to study the reversible covalent and non-covalent inhibitors of prolyl oligopeptidase (POP). We extracted kinetics spanning three orders of magnitude, including those too rapid for standard methods, and measured sub-nM binding affinities below the typical ITC limit. These results shed light on the inhibition of POP and demonstrate the general utility of ITC-based enzyme inhibition kinetic measurements.

Efficient and Rapid Mechanochemical Assembly of Platinum(II) Squares for Guanine Quadruplex Targeting.

We present a rapid and efficient method to generate a family of platinum supramolecular square complexes, including previously inaccessible targets, through the use of ball milling mechanochemistry. This one-pot, two-step process occurs in minutes and enables the synthesis of the squares [Pt4(en)4(N∩N)4][CF3SO3]8 (en= ethylenediamine, N∩N = 4,4'-bipyridine derivatives) from commercially available precursor K2PtCl4 in good to excellent yields. In contrast, solution-based assembly requires heating the reagents for weeks and gives lower yields. Mechanistic investigations into this remarkable rate acceleration revealed that solution-based assembly (refluxing for days) results in the formation of large oligomeric side-products that are difficult to break down into the desired squares. On the other hand, ball milling in the solid state is rapid and appears to involve smaller intermediates. We examined the binding of the new supramolecular squares to guanine quadruplexes, including oncogene and telomere-associated DNA and RNA sequences. Sub-micromolar binding affinities were obtained by fluorescence displacement assays (FID) and isothermal titration calorimetry (ITC), with binding preference to telomere RNA (TERRA) sequences. ITC showed a 1:1 binding stoichiometry of the metallosquare to TERRA, while the stoichiometry was more complex for telomeric quadruplex DNA and a double-stranded DNA control.

Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological interactions including molecular recognition and enzymatic catalysis. ITC-based kinetics measurements typically focus on reactions taking place over long time scales (tens of minutes or hours) in order to avoid complications due to the finite length of time needed detect heat flow in the calorimeter cell. While progress has been made toward analyzing more rapid reaction kinetics by ITC, the capabilities and limitations of this approach have not been thoroughly tested to date. Here, we report that the time resolution of commercial instruments is on the order of 0.2 s or less. We successfully performed rapid ITC kinetics assays with durations of just tens of seconds using the enzyme trypsin. This is substantially shorter than previous ITC enzyme measurements. However, we noticed that for short reaction durations, standard assumptions regarding the ITC instrument response led to significant deviations between calculated and measured ITC peak shapes. To address this issue, we developed an ITC empirical response model (ITC-ERM) that quantitatively reproduces ITC peak shapes for all reaction durations. Applying the ITC-ERM approach to another enzyme (prolyl oligopeptidase), we unexpectedly discovered non-Michaelis-Menten kinetics in short time-scale measurements that are absent in more typical long time-scale experiments and are obscured in short time-scale experiments when standard assumptions regarding the instrument response are made. This highlights the potential of ITC measurements of rapid time scale kinetics in conjunction with the ITC-ERM approach to shed new light on biological dynamics.

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus.

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)(+) with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and (15)N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.