Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

Modeling diffuse scattering with simple, physically interpretable models.

Diffuse scattering has long been proposed to probe protein dynamics relevant for biological function, and more recently, as a tool to aid structure determination. Despite recent advances in measuring and modeling this signal, the field has not been able to routinely use experimental diffuse scattering for either application. A persistent challenge has been to devise models that are sophisticated enough to robustly reproduce experimental diffuse features but remain readily interpretable from the standpoint of structural biology. This chapter presents eryx, a suite of computational tools to evaluate the primary models of disorder that have been used to analyze protein diffuse scattering. By facilitating comparative modeling, eryx aims to provide insights into the physical origins of this signal and help identify the sources of disorder that are critical for reproducing experimental features. This framework also lays the groundwork for the development of more advanced models that integrate different types of disorder without loss of interpretability.

Structural basis for functional properties of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

Skopi: a simulation package for diffractive imaging of noncrystalline biomolecules.

X-ray free-electron lasers (XFELs) have the ability to produce ultra-bright femtosecond X-ray pulses for coherent diffraction imaging of biomolecules. While the development of methods and algorithms for macromolecular crystallography is now mature, XFEL experiments involving aerosolized or solvated biomolecular samples offer new challenges in terms of both experimental design and data processing. Skopi is a simulation package that can generate single-hit diffraction images for reconstruction algorithms, multi-hit diffraction images of aggregated particles for training machine learning classifiers using labeled data, diffraction images of randomly distributed particles for fluctuation X-ray scattering algorithms, and diffraction images of reference and target particles for holographic reconstruction algorithms. Skopi is a resource to aid feasibility studies and advance the development of algorithms for noncrystalline experiments at XFEL facilities.

Montage electron tomography of vitrified specimens.

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

CryoAI: Amortized Inference of Poses for Ab Initio Reconstruction of 3D Molecular Volumes from Real Cryo-EM Images.

Cryo-electron microscopy (cryo-EM) has become a tool of fundamental importance in structural biology, helping us understand the basic building blocks of life. The algorithmic challenge of cryo-EM is to jointly estimate the unknown 3D poses and the 3D electron scattering potential of a biomolecule from millions of extremely noisy 2D images. Existing reconstruction algorithms, however, cannot easily keep pace with the rapidly growing size of cryo-EM datasets due to their high computational and memory cost. We introduce cryoAI, an ab initio reconstruction algorithm for homogeneous conformations that uses direct gradient-based optimization of particle poses and the electron scattering potential from single-particle cryo-EM data. CryoAI combines a learned encoder that predicts the poses of each particle image with a physics-based decoder to aggregate each particle image into an implicit representation of the scattering potential volume. This volume is stored in the Fourier domain for computational efficiency and leverages a modern coordinate network architecture for memory efficiency. Combined with a symmetrized loss function, this framework achieves results of a quality on par with state-of-the-art cryo-EM solvers for both simulated and experimental data, one order of magnitude faster for large datasets and with significantly lower memory requirements than existing methods.

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation.

Structure-determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure, but is constrained by the need for large, well ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular-weight threshold required for single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure-determination methods. Here, a data-processing scheme is presented that combines routines from X-ray crystallography and new algorithms that have been developed to solve structures from tomograms of nanocrystals. This pipeline handles image-processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. The tolerance of this workflow to the effects of radiation damage is also assessed. The simulations indicate a trade-off between a wider tilt range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors, but not merging errors, can be overcome with additional data sets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rß, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals.

Conformational changes drive protein function, including catalysis, allostery and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work has challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid-body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signal reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid-body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase.

The catalytic mechanisms underlying Escherichia coli alkaline phosphatase's (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein. The crystal structure of tungstate-bound alkaline phosphatase provides evidence for a covalent adduct model and further shows that the ligand adopts trigonal bipyramidal geometry, which is infrequently observed for tungstate in small molecules and other active sites but mirrors the geometry of the presumed phosphoryl transfer transition state. The AP active site is known to stabilize another oxyanion, vanadate, in trigonal bipyramidal geometry, but the extent to which binding of either ligand reproduces the energetics of the transition state cannot be deduced from structural inspection alone. To test for transition state analog behavior, we determined the relationship between catalytic activity and affinity for tungstate and vanadate for a series of 20 AP variants. Affinity and activity were highly correlated for tungstate (r(2) = 0.89) but not vanadate (r(2) = 0.23), indicating that the tungstate•AP complex may better mimic this enzyme's transition state properties. The results herein suggest that tungstate will be a valuable tool for further dissecting AP catalysis and may prove helpful in mechanistic studies of other phosphoryl transfer enzymes.

Conformational heterogeneity of the calmodulin binding interface.

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca(2+), sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca(2+)-bound regime and, thus, a candidate for pharmacological intervention.

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Alternative activation in systemic juvenile idiopathic arthritis monocytes.

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1ß after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

Distribution of circulating cells in systemic juvenile idiopathic arthritis across disease activity states.

Juvenile idiopathic arthritis (JIA) encompasses a group of chronic childhood arthritides of unknown etiology. One subtype, systemic JIA (SJIA), is characterized by a combination of arthritis and systemic inflammation. Its systemic nature suggests that clues to SJIA pathogenesis may be found in examination of peripheral blood cells. To determine the immunophenotypic profiles of circulating mononuclear cells in SJIA patients with different degrees of disease activity, we studied PBMC from 31 SJIA patients, 20 polyarticular JIA patients (similar to adult rheumatoid arthritis), and 31 age-matched controls. During SJIA disease flare, blood monocyte numbers were increased, whereas levels of myeloid dendritic cells (DC) and gammadelta T cells were reduced. At both flare and quiescence, increased levels of CD14 and CD16 were found on SJIA monocytes. Levels of CD16-DC were elevated at SJIA quiescence compared both to healthy controls and to SJIA subjects with active disease. Overall, our findings suggest dysregulation of innate immunity in SJIA and raise the possibility that quiescence represents a state of compensated inflammation.

Precarious balance: Th17 cells in host defense.

Lineage-specific responses from the effector T-cell repertoire form a critical component of adaptive immunity. The recent identification of Th17 cells-a third, distinct lineage of helper T cells-collapses the long-accepted paradigm in which Th1 and Th2 cells distinctly mediate cellular and humoral immunity, respectively. In this minireview, we discuss the involvement of the Th17 lineage during infection by extracellular bacteria, intracellular bacteria, and fungi. Emerging trends suggest that the Th17 population bridges innate and adaptive immunity to produce a robust antimicrobial inflammatory response. However, because Th17 cells mediate both host defense and pathological inflammation, elucidation of mechanisms that attenuate but do not completely abolish the Th17 response may have powerful implications for therapy.

Plasticity of T-cell phenotype and function: the T helper type 17 example.

Mature T helper type 1 (Th1) and Th2 cells antagonize the development of the opposing subset to sustain lineage-specific responses. However, the recent identification of a third distinct subset of helper T cells - the Th17 lineage - collapses the established Th1/Th2 dichotomy and raises intriguing questions about T-cell fate. In this review, we discuss the Th17 subset in the context of the effector and regulatory T-cell lineages. Initial studies suggested reciprocal developmental pathways between Th17/Th1 subsets and between Th17/regulatory T-cell subsets, and identified multiple mechanisms by which Th1 and Th2 cells antagonize the generation of Th17 cells. However, recent observations reveal the susceptibility of differentiated Th17 cells to Th1 polarization and the enhancement of Th17 memory cells by the Th1 factors interferon-gamma and T-bet. In addition, new data indicate late-stage plasticity of a subpopulation of regulatory T cells, which can be selectively induced to adopt a Th17 phenotype. Elucidating the mechanisms that undermine cross-lineage suppression and facilitate these phenotype shifts will not only clarify the flexibility of T-cell differentiation, but may also shed insight into the pathogenesis of autoimmunity and cancer. Furthermore, understanding these phenomena will be critical for the design of immunotherapy that seeks to disrupt lineage-specific T-cell responses and may suggest ways to manipulate the balance between pathogenic and regulatory lymphocytes for the restoration of homeostasis.