RESUMEN
Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aqueous mixtures of isopropanol (IPA) with water will decrease by â¼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent khydride rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational substates, explaining the decreased khydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.
Asunto(s)
2-Propanol/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/enzimología , Solventes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Cristalografía por Rayos X/métodos , Escherichia coli/química , Escherichia coli/metabolismo , Cinética , Simulación de Dinámica Molecular , Conformación Proteica/efectos de los fármacos , Electricidad Estática , Tetrahidrofolato Deshidrogenasa/química , Viscosidad , Agua/metabolismoRESUMEN
BACKGROUND: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized. METHODS: Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa. RESULTS: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation. CONCLUSIONS: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D. GENERAL SIGNIFICANCE: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.
Asunto(s)
Deshidrogenasas de Carbohidratos/química , Proteínas Fúngicas/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Ascomicetos/enzimología , Ascomicetos/genética , Calcio/química , Calcio/metabolismo , Deshidrogenasas de Carbohidratos/genética , Deshidrogenasas de Carbohidratos/metabolismo , Celulosa/química , Celulosa/metabolismo , Transporte de Electrón , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Neurospora crassa/enzimología , Neurospora crassa/genética , Oxidación-Reducción , Unión Proteica , Conformación ProteicaRESUMEN
Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Neurospora crassa/enzimología , Oxígeno/metabolismo , Polisacáridos/metabolismo , Dominio Catalítico , Oxigenasas de Función Mixta/química , Modelos Moleculares , Oxígeno/química , Polisacáridos/químicaRESUMEN
RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.
Asunto(s)
Guanosina Trifosfato/química , Neutrones , Proteínas Proto-Oncogénicas p21(ras)/química , Catálisis , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.
Asunto(s)
Cristalización/métodos , Hidrógeno/análisis , Hidrógeno/química , Difracción de Neutrones/métodos , Proteínas/química , Proteínas/ultraestructura , Simulación por Computador , Modelos Químicos , Modelos Moleculares , Conformación ProteicaRESUMEN
Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.
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Colesterol Oxidasa/química , Deuterio/química , Escherichia coli/química , Marcaje Isotópico/métodos , Colesterol Oxidasa/biosíntesis , Colesterol Oxidasa/genética , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl group via an activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Proteínas Bacterianas/química , Glucosa/química , Protones , Streptomyces/química , Isomerasas Aldosa-Cetosa/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Glucosa/análogos & derivados , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Difracción de Neutrones , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Dispersión del Ángulo Pequeño , Streptomyces/enzimología , Difracción de Rayos XRESUMEN
Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP(2). Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP(2) binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP(2) binding. The study provides the first structural view of the activated ezrin bound to PIP(2) and to F-actin.
Asunto(s)
Actinas/química , Proteínas del Citoesqueleto/química , Fosfatidilinositol 4,5-Difosfato/química , Sustitución de Aminoácidos , Proteínas del Citoesqueleto/genética , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Difracción de Neutrones , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Intercambiadores de Sodio-Hidrógeno/química , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
The first high-resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported. Neutron diffraction data extending to 1.65â Å resolution were collected from a relatively small 0.7â mm(3) PfRd crystal using 2.5â d (60â h) of beam time. The refined structure contains 371 out of 391, or 95%, of the D atoms of the protein and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolution (1.5â Å) from crystals with volume <1.0â mm(3) and with unit-cell edges <100â Å. Beamline features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2â mm focal spot at the sample position with full-width vertical and horizontal divergences of 0.5 and 0.6°, respectively. Variable short- and long-wavelength cutoff optics provide automated exchange between multiple-wavelength configurations (λmin = 2.0, 2.8, 3.3â Å to λmax = 3.0, 4.0, 4.5, â¼20â Å). These optics produce a more than 20-fold increase in the flux density at the sample and should help to enable more routine collection of high-resolution data from submillimetre-cubed crystals. Notably, the crystal used to collect these PfRd data was 5-10 times smaller than those previously reported.
Asunto(s)
Cristalografía por Rayos X/métodos , Sustancias Macromoleculares/química , Neutrones , Pyrococcus furiosus/química , Rubredoxinas/química , Proteínas Arqueales/química , Cristalografía por Rayos X/instrumentación , Enlace de Hidrógeno , Dispersión de Radiación , Difracción de Rayos XRESUMEN
Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.
Asunto(s)
Virus Sindbis/química , Virión/química , Concentración de Iones de Hidrógeno , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Dispersión del Ángulo Pequeño , Virus Sindbis/genética , Virus Sindbis/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Virión/genética , Virión/metabolismoRESUMEN
Red clover necrotic mosaic virus (RCNMV) is a 36-nm-diameter, T = 3 icosahedral plant virus with a genome that is split between two single-stranded RNA molecules of approximately 3.9 kb and 1.5 kb, as well as a 400-nucleotide degradation product. The structure of the virus capsid and its response to removing Ca(2+) and Mg(2+) was previously studied by cryo-electron microscopy (cryo-EM) (Sherman et al. J Virol 80:10395-10406, 2006) but the structure of the RNA was only partially resolved in that study. To better understand the organization of the RNA and conformational changes resulting from the removal of divalent cations, small-angle neutron scattering with contrast variation experiments were performed. The results expand upon the cryo-EM results by clearly showing that virtually all of the RNA is contained in a thin shell that is in contact with the interior domains of the viral capsid protein, and they provide new insight into changes in the RNA packing that result from removal of divalent cations.
Asunto(s)
Cationes Bivalentes/química , Conformación de Ácido Nucleico , ARN Viral/química , Tombusviridae/genética , Cationes Bivalentes/metabolismo , Modelos Biológicos , Modelos Moleculares , Nucleocápside/química , Nucleocápside/metabolismo , ARN Viral/metabolismo , Dispersión del Ángulo Pequeño , Tombusviridae/químicaRESUMEN
ARTICLES DISCUSSED: Vahdatahar, E., Junius, N., Budayova-Spano, M. Optimization of crystal growth for neutron macromolecular crystallography. Journal of Visualized Experiments. (169), e61685 (2021). Schröder, G. C., Meilleur, F. Neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures. Journal of Visualized Experiments. (166), e61903 (2020). Kelley, E. G., Nguyen, M. H. L., Marquardt, D., Maranville, B. B., Murphy, R. P. Measuring the time-evolution of nanoscale materials with stopped-flow and small-angle neutron scattering. Journal of Visualized Experiments. (174), e62873 (2021). Bilheux, H. Z. et al. Neutron radiography and computed tomography of biological systems at the Oak Ridge National Laboratory's high flux isotope reactor. Journal of Visualized Experiments. (171), e61688 (2021). Stingaciu, L.-R. Study of protein dynamics via neutron spin echo spectroscopy. Journal of Visualized Experiments. (182), e61862 (2022). Kumarage, T., Nguyen, J., Ashkar, R. Neutron spin echo spectroscopy as a unique probe for lipid membrane dynamics and membrane-protein interactions. Journal of Visualized Experiments. (171), e62396 (2021).
Asunto(s)
Disciplinas de las Ciencias Biológicas , Cristalización , Recolección de Datos , Hidrógeno , NeutronesRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state refined against X-ray and neutron data at 2.1 and 2.8â Å resolution, respectively, is presented. The results provide an experimental determination of the protonation states of the copper(II)-coordinating residues and second-shell residues in LsAA9_A, paving the way for future neutron crystallographic studies of LPMO-carbohydrate complexes.
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Cobre , Polisacáridos , Rayos X , Temperatura , Cristalografía por Rayos X , Polisacáridos/químicaRESUMEN
Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1-110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region's impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%-97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.
RESUMEN
Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.
Asunto(s)
Hidrógeno/análisis , Difracción de Neutrones/métodos , Proteínas/química , Hidrógeno/química , Modelos Moleculares , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory calculations indicate that both superoxo and hydroperoxo active site states are stable. The hydroperoxo formed is potentially an early LPMO catalytic reaction intermediate or the first step in the mechanism of hydrogen peroxide formation in the absence of substrate. We observe that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by molecular dynamics and mining minima free energy calculations we establish that the conserved second-shell His161 in MtPMO3* displays conformational flexibility in solution and that this flexibility is also observed, though to a lesser extent, in His157 of NcLPMO9D. The imidazolate form of His157 observed in our structure following oxygen intermediate protonation can be attributed to abolished His157 flexibility due steric hindrance in the crystal as well as the solvent-occluded active site environment due to crystal packing. A neutron crystal structure of NcLPMO9D at low pH further supports occlusion of the active site since His157 remains singly protonated even at acidic conditions.
RESUMEN
When strands of DNA encapsulate silver clusters, supramolecular optical chromophores develop. However, how a particular structure endows a specific spectrum remains poorly understood. Here, we used neutron diffraction to map protonation in (A2C4)2-Ag8, a green-emitting fluorophore with a "Big Dipper" arrangement of silvers. The DNA host has two substructures with distinct protonation patterns. Three cytosines from each strand collectively chelate handle-like array of three silvers, and calorimetry studies suggest Ag+ cross-links. The twisted cytosines are further joined by hydrogen bonds from fully protonated amines. The adenines and their neighboring cytosine from each strand anchor a dipper-like group of five silvers via their deprotonated endo- and exocyclic nitrogens. Typically, exocyclic amines are strongly basic, so their acidification and deprotonation in (A2C4)2-Ag8 suggest that silvers perturb the electron distribution in the aromatic nucleobases. The different protonation states in (A2C4)2-Ag8 suggest that atomic level structures can pinpoint how to control and tune the electronic spectra of these nanoscale chromophores.
Asunto(s)
ADNRESUMEN
The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.
RESUMEN
The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.
RESUMEN
The recently discovered lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes capable of degrading polysaccharide substrates oxidatively. The generally accepted first step in the LPMO reaction is the reduction of the active-site metal ion from Cu2+ to Cu+. Here we have used a systematic diffraction data collection method to monitor structural changes in two AA9 LPMOs, one from Lentinus similis (LsAA9_A) and one from Thermoascus auranti-acus (TaAA9_A), as the active-site Cu is photoreduced in the X-ray beam. For LsAA9_A, the protein produced in two different recombinant systems was crystallized to probe the effect of post-translational modifications and different crystallization conditions on the active site and metal photoreduction. We can recommend that crystallographic studies of AA9 LPMOs wishing to address the Cu2+ form use a total X-ray dose below 3 × 104â Gy, while the Cu+ form can be attained using 1 × 106â Gy. In all cases, we observe the transition from a hexa-coordinated Cu site with two solvent-facing ligands to a T-shaped geometry with no exogenous ligands, and a clear increase of the θ2 parameter and a decrease of the θ3 parameter by averages of 9.2° and 8.4°, respectively, but also a slight increase in θT. Thus, the θ2 and θ3 parameters are helpful diagnostics for the oxidation state of the metal in a His-brace protein. On binding of cello-oligosaccharides to LsAA9_A, regardless of the production source, the θT parameter increases, making the Cu site less planar, while the active-site Tyr-Cu distance decreases reproducibly for the Cu2+ form. Thus, the θT increase found on copper reduction may bring LsAA9_A closer to an oligosaccharide-bound state and contribute to the observed higher affinity of reduced LsAA9_A for cellulosic substrates.