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Bromodomain-containing protein 4 (BRD4) recognizes the acetylated lysine of histone H4 via its bromodomains, leading to the recruitment of positive transcription elongation factor b. Small molecules that inhibit BRD4 have potential as anticancer agents by leading to the downregulation of specific oncogenes. Using X-ray crystallographic screening, we identified the BRD4 inhibitory activity of isoliquiritigenin (ISL), a natural chalcone found in licorice. Structural analysis revealed that ISL bound to BRD4 with a novel binding mode and squeezed out one of the six conserved water molecules that form a strong hydrogen bond network. The thermodynamic analysis revealed that the binding of ISL is enthalpy driven, suggesting that strong hydrogen bonds would compensate for the desolvation penalty. Neutron protein crystallography further suggested that the favorable binding enthalpy originates from the stabilization and optimization of the hydrogen bond network of the conserved water molecules. Here, we describe the novelty and potential of ISL as a template for new BRD4 inhibitors.

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IX mimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IX mimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

Using lysozyme as a crystallization model, existing intermediate clusters and aggregates have been previously identified as fractal systems using light scattering techniques. However, this has not been confirmed with neutron or X-ray scattering directly. In this work, we attempt to deepen our knowledge of the role of the fractal clusters during the crystallization process by following the evolution of the fractal dimension df from the early stage of the nucleation process. Indeed, three different scattering techniques have been used simultaneously on the same sample: dynamic light scattering, small-angle neutron scattering, and static light scattering. We focused on the optimal batch crystallization condition in order to obtain large crystals (30 mg/mL lysozyme concentration and 3 wt % sodium chloride at pD 4.75 at 298 K). The selected temperature reduces the nucleation speed allowing us to investigate in detail the very early stage of the crystallization process. A direct temporal change of the fractal d...

The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.

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.

Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate–protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A ofRhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm3) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement usingphenix.refineshowed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein–carbohydrate binding interactions.

The use of cryocooling in neutron diffraction has been hampered by several technical challenges, such as the need for specialized equipment and techniques. This article reports the recent development and deployment of equipment and strategies that allow routine neutron data collection on cryocooled crystals using off-the-shelf components. This system has several advantages compared to a closed displex cooling system, such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at the FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure, a full neutron data set was collected on the BIODIFF instrument on a Toho-1 β-lactamase structure at 100 K.

The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with 11B, as 10B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction. Background: Antibiotic resistance from extended-spectrum β-lactamases (ESBLs) makes infections more dangerous and difficult to treat. Results: Neutron and x-ray crystal structures were determined for an ESBL in complex with an acylation transition state analog. Conclusion: Glu-166 is implicated as the general base in the acylation reaction. Significance: Understanding the catalytic mechanism of β-lactamases will lead to improved antibiotics and β-lactamase inhibitors.

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS–risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (∼3.5 mm3) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported.

Intramolecular charge transfer in aminobenzonitrile (DIABN) single crystals is studied by a combination of ultrafast techniques. The intramolecular dynamics is probed by time-resolved spectroscopy in the UV/Vis and mid-IR. The intermolecular structural response is investigated by transient X-ray scattering and transmission experiments. A theoretical analysis shows that diffuse scattering and dipole solvation determine the X-ray data. The observed dynamics results from charge transfer reaction leading to strong local changes of molecular dipole moments and related structural rearrangements in the crystal.

The photodynamics of single crystals of 4-(diisopropylamino)benzonitrile (DIABN) have been studied by femtosecond UV/Vis and mid-IR absorption spectroscopy. The observed spectroscopic response, especially in the UV/Vis, strongly depends on the optical excitation conditions. For the excitation light in resonance with the absorption of DIABN, broad and structureless transient spectra are observed, which decay non-exponentially within pico- to nanoseconds. For off-resonance excitation in the very red wing of the absorption band a stimulated emission signal is detected, which decays in ~10 ps matching the known behavior of the non-interacting chromophore in solution. The 10 ps decay is due to an intramolecular charge transfer process which is supported by femtosecond IR spectroscopy. The observed dependence on the excitation wavelength is assigned to a change in the density of excited chromophores and therefore bears relevance for time resolved X-ray diffraction experiments.

Ultrafast IR-pump IR-probe spectroscopy is used to study the relaxation dynamics of propionic acid dimers. The spectral changes in the regions of CO-stretching at ∼1700 cm −1 , OH and CH-bending modes around 1400 cm −1 are investigated after excitation in the range of the CH- and CO-stretching modes. A strong coupling of the OH-stretch vibration to CO-stretching and OH/CH-bending modes is found. The intramolecular redistribution of vibrational energy takes place on a 1 ps time-scale. A further cooling process and energy transfer to the surrounding occur with ∼12 ps. The dynamics depend on the wavelength of the IR-excitation and point to a transient breaking of one of the two H-bonds of the propionic acid dimers.

The prototype operation of an ultrafast write-readout-erase-readout cycle of an all-optical system based on photochromic indolylfulgides is demonstrated. In the employed dye the molecular structure is switched using light of different wavelengths between two thermally stable states to allow binary encoding of information. Non-destructive readout of the bit states using infrared light completes the scheme of an all-optical memory. For ultrafast operation femtosecond light pulses are applied and it is demonstrated that two consecutive write/erase processes separated by less than 4 ps still allow the defined readout of the bit state. The short time between the write/erase and readout actions demonstrates that an all-optical data storage system based on indolylfulgides may be operated at memory clock rates that exceed 250 GHz.

Femtosecond time-resolved infrared spectroscopy was used to study the formation of cyclobutane dimers in the all-thymine oligodeoxynucleotide (dT)18by ultraviolet light at 272 nanometers. The appearance of marker bands in the time-resolved spectra indicates that the dimers are fully formed ∼1 picosecond after ultraviolet excitation. The ultrafast appearance of this mutagenic photolesion points to an excited-state reaction that is approximately barrierless for bases that are properly oriented at the instant of light absorption. The low quantum yield of this photoreaction is proposed to result from infrequent conformational states in the unexcited polymer, revealing a strong link between conformation before light absorption and photodamage.

The photo-induced trans–cis isomerization of the azobenzene derivative 4-nitro-4 0 -(dimethylamino)azobenzene in polar solution was studied by femtosecond UV/Vis and IR spectroscopy. The UV/Vis experiment reveals two excited state processes; the slower one (1 ps) is the internal conversion to the ground state. The ensuing spectral changes point to vibrational cooling of the nascent cis product and the recovered trans isomer. Judging from the UV/Vis experiment this � 5 ps process seems to terminate the isomerization. The internal conversion and the cooling also find their manifestation in the IR experiment. In addition slower spectral changes lasting until � 50 ps are detected. These slow IR responses are compared with the behavior of a non-isomerizing analogue, para-N,N-dimethyl-nitroaniline. An origin for this discrepancy is suggested and potential molecular processes causing the slow IR response are discussed. 2007 Elsevier B.V. All rights reserved.

The photo-induced ring-opening reaction C → E of a photochromic indolyl-fulgimide is investigated by sub-picosecond pump–probe spectroscopy in the visible and infrared spectral range. The thermally stable C-isomer shows a pronounced absorption in the visible spectral range without spectral overlap with the absorption of the E- and Z-isomer. The comparison of infrared and visible transient absorption data allows to assign the observed exponential 2.1 ps dynamics to the ring-opening reaction and the non-exponential 10–40 ps component to the cooling of the hot molecules. The transient absorption spectra taken 80 ps after photo-excitation are identical with the steady state difference spectra.

Femtosecond IR and Raman techniques are compared with respect to their abilities to monitor vibrational relaxation following ultrafast internal conversion. The study was performed on para -nitroaniline which undergoes sub-picosecond internal conversion to the ground state populating a variety of vibrational modes. The NO 2 stretch vibration at 1315 cm −1 responds to the excitation by a strong red shift, visible in transient IR and Raman experiments. It is due to the off-diagonal anharmonic coupling with other modes. The population of the NO 2 stretch mode itself can only be observed in the (anti-Stokes) Raman spectrum, whereas IR spectroscopy is superior in detecting the coupling induced shift.

We study a monocyclic peptide called cAPB, whose conformations are light switchable due to the covalent integration of an azobenzene dye. Molecular dynamics (MD) simulations using the CHARMM22 force field and its CMAP extension serve us to sample the two distinct conformational ensembles of cAPB, which belong to the cis and trans isomers of the dye, at room temperature. For gaining sufficient statistics we apply a novel replica exchange technique. We find that the well-known NMR distance restraints are much better described by CMAP than by CHARMM22. In cAPB, the ultrafast cis/trans photoisomerization of the dye elicits a relaxation dynamics of the peptide backbone. Experimentally, we probe this relaxation at picosecond time resolution by IR spectroscopy in the amide I range up to 3 ns after the UV/vis pump flash. We interpret the spectroscopically identified decay kinetics using ensembles of non-equilibrium MD simulations, which provide kinetic data on conformational transitions well matching the observed kinetics. Whereas spectroscopy solely indicates that the relaxation toward the equilibrium trans ensemble is by no means complete after 3 ns, the 20 ns MD simulations of the process predict, independently of the applied force field, that the final relaxation into the trans-ensemble proceeds on a time scale of 23 ns. Overall our explicit solvent simulations cover more than 6 micros.