Mechanism of allosteric inhibition of human p97/VCP ATPase and its disease mutant by triazole inhibitors.

Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been demonstrated to match the efficacy of CB-5083, an ATP-competitive inhibitor, in cellular models. However, the mechanism is not well understood. This study systematically investigates the structures of new triazole inhibitors bound to  both wild-type and disease mutant forms of p97 and measures their effects on function. These inhibitors bind at the interface of the D1 and D2 domains of each p97 subunit, shifting surrounding helices and altering the loop structures near the C-terminal α2 G helix to modulate domain-domain communications. A key structural moiety of the inhibitor affects the rotameric conformations of interacting side chains, indirectly modulating the N-terminal domain conformation in p97 R155H mutant. The differential effects of inhibitor binding to wild-type and mutant p97 provide insights into drug design with enhanced specificity, particularly for oncology applications.

The Pixel Anomaly Detection Tool: a user-friendly GUI for classifying detector frames using machine-learning approaches.

Data collection at X-ray free electron lasers has particular experimental challenges, such as continuous sample delivery or the use of novel ultrafast high-dynamic-range gain-switching X-ray detectors. This can result in a multitude of data artefacts, which can be detrimental to accurately determining structure-factor amplitudes for serial crystallography or single-particle imaging experiments. Here, a new data-classification tool is reported that offers a variety of machine-learning algorithms to sort data trained either on manual data sorting by the user or by profile fitting the intensity distribution on the detector based on the experiment. This is integrated into an easy-to-use graphical user interface, specifically designed to support the detectors, file formats and software available at most X-ray free electron laser facilities. The highly modular design makes the tool easily expandable to comply with other X-ray sources and detectors, and the supervised learning approach enables even the novice user to sort data containing unwanted artefacts or perform routine data-analysis tasks such as hit finding during an experiment, without needing to write code.

Review of serial femtosecond crystallography including the COVID-19 pandemic impact and future outlook.

Serial femtosecond crystallography (SFX) revolutionized macromolecular crystallography over the past decade by enabling the collection of X-ray diffraction data from nano- or micrometer sized crystals while outrunning structure-altering radiation damage effects at room temperature. The serial manner of data collection from millions of individual crystals coupled with the femtosecond duration of the ultrabright X-ray pulses enables time-resolved studies of macromolecules under near-physiological conditions to unprecedented temporal resolution. In 2020 the rapid spread of the coronavirus SARS-CoV-2 resulted in a global pandemic of coronavirus disease-2019. This led to a shift in how serial femtosecond experiments were performed, along with rapid funding and free electron laser beamtime availability dedicated to SARS-CoV-2-related studies. This review outlines the current state of SFX research, the milestones that were achieved, the impact of the global pandemic on this field as well as an outlook into exciting future directions.

Heterogeneity in M. tuberculosis ß-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme.

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu B ) and a heme a 3 iron, where O 2 binds and is reduced to water. CO is a versatile O 2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (Cu B -CO), where CO is bound to Cu B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the Cu B and the heme a 3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

Heterogeneity in the M. tuberculosis ß-Lactamase Inhibition by Sulbactam.

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) 1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals 6 . Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating 7-9 , cooperativity, induced fit 10,11 and conformational selection 11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans- enamine. This was made possible in part by the application of the singular value decomposition 14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Native Deglycosylation and Size Exclusion Chromatography of Viral Chemokine Binding Proteins for Structural Discovery.

Structural discovery of viral chemokine binding proteins can provide valuable information on the binding domains and protein-protein interfaces (PPI) of these immunologically relevant proteins. Protein expression in mammalian cells produces high-quality protein compared to other expression methods; however, because structural discovery methods such as cryo-EM-based single particle analysis (SPA) and x-ray crystallography use methods which combine data from many individual proteins, these demand a highly monodispersed sample composed of protein with ordered structure. These techniques are often incompatible with flexible glycosyl groups commonly present on proteins produced by mammalian cells and require deglycosylation to enable observation of the conserved tertiary structure beneath these variable, flexible, glycans. Using the Myxoma viral protein M-T7 as a test case, we discuss considerations and preliminary bioinformatic analysis for approaching structural discovery using freely accessible sequence and structure databases to maximize success and guide experiments. We describe a simple deglycosylation optimization protocol utilizing Endo H followed by size exclusion chromatography (SEC) based purification to produce and validate protein suitable for structural discovery. Considerations such as protein concentration and volumes required for crystallography and negative stain electron microscopy are discussed as well as grid blotting techniques for negative stain experiments to validate protein quality.

Regulation of light energy conversion between linear and cyclic electron flow within photosystem II controlled by the plastoquinone/quinol redox poise.

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.

Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis.

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (~6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a ß-barrel domain consistent with alphafold2's prediction of an eight stranded ß-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal ß-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal ß-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor.

Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.

Serial macromolecular crystallography at ALBA Synchrotron Light Source. Erratum.

A revised version of Table 2 of Martin-Garcia et al. [J. Synchrotron Rad. (2022). 29, 896-907] is provided.

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma.

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic-Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

A novel solution for controlling hardware components of accelerators and beamlines.

A novel approach to the remote-control system for the compact multi-crystal energy-dispersive spectrometer for X-ray emission spectroscopy (XES) applications has been developed. This new approach is based on asynchronous communication between software components and on reactive design principles. In this paper, the challenges faced, their solutions, as well as the implementation and future development prospects are identified. The main motivation of this work was the development of a new holistic communication protocol that can be implemented to control various hardware components allowing both independent operation and easy integration into different SCADA systems.

Serial macromolecular crystallography at ALBA Synchrotron Light Source.

The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 µm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 µm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.

Why Did Nature Choose Manganese over Cobalt to Make Oxygen Photosynthetically on the Earth?

All contemporary oxygenic phototrophs─from primitive cyanobacteria to complex multicellular plants─split water using a single invariant cluster comprising Mn4CaO5 (the water oxidation catalyst) as the catalyst within photosystem II, the universal oxygenic reaction center of natural photosynthesis. This cluster is unstable outside of PSII and can be reconstituted, both in vivo and in vitro, using elemental aqueous ions and light, via photoassembly. Here, we demonstrate the first functional substitution of manganese in any oxygenic reaction center by in vitro photoassembly. Following complete removal of inorganic cofactors from cyanobacterial photosystem II microcrystal (PSIIX), photoassembly with free cobalt (Co2+), calcium (Ca2+), and water (OH-) restores O2 evolution activity. Photoassembly occurs at least threefold faster using Co2+ versus Mn2+ due to a higher quantum yield for PSIIX-mediated charge separation (P*): Co2+ → P* → Co3+QA-. However, this kinetic preference for Co2+ over native Mn2+ during photoassembly is offset by significantly poorer catalytic activity (∼25% of the activity with Mn2+) and ∼3- to 30-fold faster photoinactivation rate. The resulting reconstituted Co-PSIIX oxidizes water by the standard four-flash photocycle, although they produce 4-fold less O2 per PSII, suggested to arise from faster charge recombination (Co3+QA ← Co4+QA-) in the catalytic cycle. The faster photoinactivation of reconstituted Co-PSIIX occurs under anaerobic conditions during the catalytic cycle, suggesting direct photodamage without the involvement of O2. Manganese offers two advantages for oxygenic phototrophs, which may explain its exclusive retention throughout Darwinian evolution: significantly slower charge recombination (Mn3+QA ← Mn4+QA-) permits more water oxidation at low and fluctuating solar irradiation (greater net energy conversion) and much greater tolerance to photodamage at high light intensities (Mn4+ is less oxidizing than Co4+). Future work to identify the chemical nature of the intermediates will be needed for further interpretation.

Electrochemically Driven Photosynthetic Electron Transport in Cyanobacteria Lacking Photosystem II.

Light-activated photosystem II (PSII) carries out the critical step of splitting water in photosynthesis. However, PSII is susceptible to light-induced damage. Here, results are presented from a novel microbial electro-photosynthetic system (MEPS) that uses redox mediators in conjunction with an electrode to drive electron transport in live Synechocystis (ΔpsbB) cells lacking PSII. MEPS-generated, light-dependent current increased with light intensity up to 2050 µmol photons m-2 s-1, which yielded a delivery rate of 113 µmol electrons h-1 mg-chl-1 and an average current density of 150 A m-2 s-1 mg-chl-1. P700+ re-reduction kinetics demonstrated that initial rates exceeded wildtype PSII-driven electron delivery. The electron delivery occurs ahead of the cytochrome b6f complex to enable both NADPH and ATP production. This work demonstrates an electrochemical system that can drive photosynthetic electron transport, provides a platform for photosynthetic foundational studies, and has the potential for improving photosynthetic performance at high light intensities.