Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD=pH+0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers.

A cellulose synthesis complex with a "rosette" shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP(+). The neutron data were collected to 2.0-Å resolution using a 3.6-mm(3) crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.

Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT.

To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca(2+) ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

Long-Range Electrostatics-Induced Two-Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site.

Neutron crystallography was used to directly locate two protons before and after a pH-induced two-proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low-pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.

Determination of cellulose crystallinity from powder diffraction diagrams.

One-dimensional (1D) (spherically averaged) powder diffraction diagrams are commonly used to determine the degree of cellulose crystallinity in biomass samples. Here, it is shown using molecular modeling how disorder in cellulose fibrils can lead to considerable uncertainty in conclusions drawn concerning crystallinity based on 1D powder diffraction data alone. For example, cellulose microfibrils that contain both crystalline and noncrystalline segments can lead to powder diffraction diagrams lacking identifiable peaks, while microfibrils without any crystalline segments can lead to such peaks. This leads to false positives, that is, assigning disordered cellulose as crystalline, and false negatives, that is, categorizing fibrils with crystalline segments as amorphous. The reliable determination of the fraction of crystallinity in any given biomass sample will require a more sophisticated approach combining detailed experiment and simulation.

Why genetic modification of lignin leads to low-recalcitrance biomass.

Genetic modification of plants via down-regulation of cinnamyl alcohol dehydrogenase leads to incorporation of aldehyde groups in the lignin polymer. The resulting lignocellulosic biomass has increased bioethanol yield. However, a molecular-scale explanation of this finding is currently lacking. Here, we perform molecular dynamics simulation of the copolymer with hemicellulose of wild type and the genetically modified lignin, in aqueous solution. We find that the non-covalent association with hemicellulose of lignin containing aldehyde groups is reduced compared to the wild-type. This phase separation may increase the cell wall porosity in the mutant plants, thus explaining their easier deconstruction to biofuels. The thermodynamic origin of the reduced lignin-hemicellulose association is found to be a more favorable self-interaction energy and less favorable interaction with hemicellulose for the mutant lignin. Furthermore, reduced hydration water density fluctuations are found for the mutant lignin, implying a more hydrophobic lignin surface. The results provide a detailed description of how aldehyde incorporation makes lignin more hydrophobic and reduces its association with hemicellulose, thus suggesting that increased lignin hydrophobicity may be an optimal characteristic required for improved biofuel production.

Direct observation of hydrogen atom dynamics and interactions by ultrahigh resolution neutron protein crystallography.

The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out of 315, or 94.9%, of the hydrogen atom positions in the protein have been experimentally derived and resolved through nuclear density maps. A number of unconventional interactions are clearly defined, including a potential O─H…π interaction between a water molecule and the aromatic ring of residue Y44, as well as a number of potential C─H…O hydrogen bonds. Hydrogen bonding networks that are ambiguous in the 0.85 Å ultrahigh resolution X-ray structure can be resolved by accurate orientation of water molecules. Furthermore, the high resolution of the reported structure has allowed for the anisotropic description of 36 deuterium atoms in the protein. The visibility of hydrogen and deuterium atoms in the nuclear density maps is discussed in relation to the resolution of the neutron data.

Metal-free cAMP-dependent protein kinase can catalyze phosphoryl transfer.

X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na(+) or K(+) coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing (32)P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis.

X-ray crystallographic studies of family 11 xylanase Michaelis and product complexes: implications for the catalytic mechanism.

Xylanases catalyze the hydrolysis of plant hemicellulose xylan into oligosaccharides by cleaving the main-chain glycosidic linkages connecting xylose subunits. To study ligand binding and to understand how the pH constrains the activity of the enzyme, variants of the Trichoderma reesei xylanase were designed to either abolish its activity (E177Q) or to change its pH optimum (N44H). An E177Q-xylohexaose complex structure was obtained at 1.15 Šresolution which represents a pseudo-Michaelis complex and confirmed the conformational movement of the thumb region owing to ligand binding. Co-crystallization of N44H with xylohexaose resulted in a hydrolyzed xylotriose bound in the active site. Co-crystallization of the wild-type enzyme with xylopentaose trapped an aglycone xylotriose and a transglycosylated glycone product. Replacing amino acids near Glu177 decreased the xylanase activity but increased the relative activity at alkaline pH. The substrate distortion in the E177Q-xylohexaose structure expands the possible conformational itinerary of this xylose ring during the enzyme-catalyzed xylan-hydrolysis reaction.

Hydration control of the mechanical and dynamical properties of cellulose.

The mechanical and dynamical properties of cellulose, the most abundant biomolecule on earth, are essential for its function in plant cell walls and advanced biomaterials. Cellulose is almost always found in a hydrated state, and it is therefore important to understand how hydration influences its dynamics and mechanics. Here, the nanosecond-time scale dynamics of cellulose is characterized using dynamic neutron scattering experiments and molecular dynamics (MD) simulation. The experiments reveal that hydrated samples exhibit a higher average mean-square displacement above ∼240 K. The MD simulation reveals that the fluctuations of the surface hydroxymethyl atoms determine the experimental temperature and hydration dependence. The increase in the conformational disorder of the surface hydroxymethyl groups with temperature follows the cellulose persistence length, suggesting a coupling between structural and mechanical properties of the biopolymer. In the MD simulation, 20% hydrated cellulose is more rigid than the dry form, due to more closely packed cellulose chains and water molecules bridging cellulose monomers with hydrogen bonds. This finding may have implications for understanding the origin of strength and rigidity of secondary plant cell walls. The detailed characterization obtained here describes how hydration-dependent increased fluctuations and hydroxymethyl disorder at the cellulose surface lead to enhancement of the rigidity of this important biomolecule.

Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase: an X-ray crystallographic study of complexes with various metals and peptide substrate SP20.

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg(2+), Ca(2+), Sr(2+), and Ba(2+) metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg(2+), nonhydrolyzable AMP-PCP (ß,γ-methyleneadenosine 5'-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants.

Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 Å).

Seeing the chemistry in biology with neutron crystallography.

New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible in most X-ray crystal structures, become visible with neutrons. Using X-rays allows one to see structure, while neutrons allow one to reveal the chemistry inherent in these macromolecular structures. A number of surprising and sometimes controversial results have emerged; because it is difficult to see or predict hydrogen atoms in X-ray structures, when they are seen by neutrons they can be in unexpected locations with important chemical and biological consequences. Here we describe examples of chemistry seen with neutrons for the first time in biological macromolecules over the past few years.

Trustworthy Data and AI Environments for Clinical Prediction: Application to Crisis-Risk in People With Depression.

Depression is a common mental health condition that often occurs in association with other chronic illnesses, and varies considerably in severity. Electronic Health Records (EHRs) contain rich information about a patient's medical history and can be used to train, test and maintain predictive models to support and improve patient care. This work evaluated the feasibility of implementing an environment for predicting mental health crisis among people living with depression based on both structured and unstructured EHRs. A large EHR from a mental health provider, Mersey Care, was pseudonymised and ingested into the Natural Language Processing (NLP) platform CogStack, allowing text content in binary clinical notes to be extracted. All unstructured clinical notes and summaries were semantically annotated by MedCAT and BioYODIE NLP services. Cases of crisis in patients with depression were then identified. Random forest models, gradient boosting trees, and Long Short-Term Memory (LSTM) networks, with varying feature arrangement, were trained to predict the occurrence of crisis. The results showed that all the prediction models can use a combination of structured and unstructured EHR information to predict crisis in patients with depression with good and useful accuracy. The LSTM network that was trained on a modified dataset with only 1000 most-important features from the random forest model with temporality showed the best performance with a mean AUC of 0.901 and a standard deviation of 0.006 using a training dataset and a mean AUC of 0.810 and 0.01 using a hold-out test dataset. Comparing the results from the technical evaluation with the views of psychiatrists shows that there are now opportunities to refine and integrate such prediction models into pragmatic point-of-care clinical decision support tools for supporting mental healthcare delivery.

Inhibition of D-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography.

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity.

Post-translational protein phosphorylation by protein kinase A (PKA) is a ubiquitous signalling mechanism which regulates many cellular processes. A low-temperature X-ray structure of the ternary complex of the PKA catalytic subunit (PKAc) with ATP and a 20-residue peptidic inhibitor (IP20) at the physiological Mg(2+) concentration of ∼0.5 mM (LT PKA-MgATP-IP20) revealed a single metal ion in the active site. The lack of a second metal in LT PKA-MgATP-IP20 renders the ß- and γ-phosphoryl groups of ATP very flexible, with high thermal B factors. Thus, the second metal is crucial for tight positioning of the terminal phosphoryl group for transfer to a substrate, as demonstrated by comparison of the former structure with that of the LT PKA-Mg(2)ATP-IP20 complex obtained at high Mg(2+) concentration. In addition to its kinase activity, PKAc is also able to slowly catalyze the hydrolysis of ATP using a water molecule as a substrate. It was found that ATP can be readily and completely hydrolyzed to ADP and a free phosphate ion in the crystals of the ternary complex PKA-Mg(2)ATP-IP20 by X-ray irradiation at room temperature. The cleavage of ATP may be aided by X-ray-generated free hydroxyl radicals, a very reactive chemical species, which move rapidly through the crystal at room temperature. The phosphate anion is clearly visible in the electron-density maps; it remains in the active site but slides about 2 Šfrom its position in ATP towards Ala21 of IP20, which mimics the phosphorylation site. The phosphate thus pushes the peptidic inhibitor away from the product ADP, while resulting in dramatic conformational changes of the terminal residues 24 and 25 of IP20. X-ray structures of PKAc in complex with the nonhydrolysable ATP analogue AMP-PNP at both room and low temperature demonstrated no temperature effects on the conformation and position of IP20.

Direct determination of the hydrogen bonding arrangement in anhydrous ß-chitin by neutron fiber diffraction.

The hydrogen bonding arrangement in anhydrous ß-chitin, a homopolymer of N-acetylglucosamine, was directly determined by neutron fiber diffraction. Data were collected from a sample prepared from the bathophilous tubeworm Lamellibrachia satsuma in which all labile hydrogen atoms had been replaced by deuterium. Initial positions of deuterium atoms on hydroxyl and acetamide groups were directly located in Fourier maps synthesized using phases calculated from the X-ray structure and amplitudes measured from the neutron data. The hydrogen bond arrangement in the refined structure is in general agreement with predictions based on the X-ray structure: O3 donates a hydrogen bond to the O5 ring oxygen atom of a neighboring residue in the same chain; N2 and O6 donate hydrogen bonds to the same carbonyl oxygen O7 of an adjacent chain. The intramolecular O3···O5 hydrogen bond has the most energetically favorable geometry with a hydrogen to acceptor distance of 1.77 Å and a hydrogen bond angle of 171°.

Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction.

Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Šresolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a=106.11, b=95.46, c=113.68 Å, α=γ=90.0, ß=98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Šresolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure-function and mechanism studies of IPPase.

Room-temperature ultrahigh-resolution time-of-flight neutron and X-ray diffraction studies of H/D-exchanged crambin.

The room-temperature (RT) X-ray structure of H/D-exchanged crambin is reported at 0.85 Å resolution. As one of the very few proteins refined with anisotropic atomic displacement parameters at two temperatures, the dynamics of atoms in the RT and 100 K structures are compared. Neutron diffraction data from an H/D-exchanged crambin crystal collected at the Protein Crystallography Station (PCS) showed diffraction beyond 1.1 Å resolution. This is the highest resolution neutron diffraction reported to date for a protein crystal and will reveal important details of the anisotropic motions of H and D atoms in protein structures.