Cooperative binding of inhaled anesthetics and ATP to firefly luciferase

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions. In the presence of ATP/Mg2+ and at room temperature, the anesthetics produce considerable stabilization with a negative DeltaH, indicating population of a conformer with a specific anesthetic binding site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets.     

Analysis of xenon binding to photosystem II by X-ray crystallography

In order to investigate oxygen binding and hydrophobic cavities in photosystem II (PSII), we have introduced xenon under pressure into crystals of PSII isolated from Thermosynechococcus elongatus and used X-ray anomalous diffraction analyses to identify the xenon sites in the complex. Under the conditions employed, 25 Xe-binding sites were identified in each monomer of the dimeric PSII complex. The majority of these were distributed within the membrane spanning portion of the complex with no obvious correlation with the previously proposed oxygen channels. One binding site was located close to the haem of cytochrome b559 in a position analogous to a Xe-binding site of myoglobin. The only Xe-binding site not associated with the intrinsic subunits of PSII was within the hydrophobic core of the PsbO protein.     

Structures of proteins and cofactors: X-ray crystallography

Protein crystallography is the predominately used technique for the determination of the three-dimensional structures of proteins and other macromolecules. In this article, the methodology utilized for the measurement and analysis of the diffraction data from crystals is briefly reviewed. As examples of both the usefulness and difficulties of this technique, the determination of the structures of several photosynthetic pigment–protein complexes is described, namely, the reaction center from purple bacteria, photosystem I and photosystem II from cyanobacteria, the light-harvesting complex II from purple bacteria, and the FMO protein from green bacteria.     

X-ray crystallography of chemical compounds

AimsAccurate knowledge of molecular structure is a prerequisite for rational drug design. This review examines the role of X-ray crystallography in providing the required structural information and advances in the field of X-ray crystallography that enhance or expand its role.Main methodsX-ray crystallography of new drugs candidates and intermediates can provide valuable information of new syntheses and parameters for quantitative structure activity relationships (QSAR).Key findingsCrystallographic studies play a vital role in many disciplines including materials science, chemistry, pharmacology, and molecular biology. X-ray crystallography is the most comprehensive technique available to determine molecular structure. A requirement for the high accuracy of crystallographic structures is that a ‘good crystal’ must be found, and this is often the rate-limiting step. In the past three decades developments in detectors, increases in computer power, and powerful graphics capabilities have contributed to a dramatic increase in the number of materials characterized by X-ray crystallography. More recently the advent of high-throughput crystallization techniques has enhanced our ability to produce that one good crystal required for crystallographic analysis.SignificanceContinuing advances in all phases of a crystallographic study have expanded the ranges of samples which can be analyzes by X-ray crystallography to include larger molecules, smaller or weakly diffracting crystals, and twinned crystals.     

The three-dimensional structure of beta2 microglobulin: results from X-ray crystallography

beta2-microglobulin, the light chain component of the major histocompatibility complex I, is involved in the development of DRA, an amyloid deposition disease occurring in man. Specifically, the beta2-microglobulin component, dissociated form the complex heavy chain, gives rise to amyloidogenic deposits in the joints of patients exposed to long dialysis periods. beta2-microglobulin three-dimensional structure is based on an antiparallel beta-barrel fold, with immunoglobulin domain topology, displaying structural flexibility in the crystal and NMR structures so fare determined. The structural bases of amyloidogenic potential in beta2-microglobulin can be related to local unfolding, to the tendency to aggregate laterally through non-compensated beta-strands, and partly also to its trend towards N-terminal proteolytic degradation. Such trends emerge quite clearly from inspection of a limited number of crystal structures of beta2-microglobulin as an isolated chain, separated form the major histocompatibility complex I heavy chain.     

X-ray crystallography of TRP channels

Transient receptor potential (TRP) ion channels are molecular sensors of a large variety of stimuli including temperature, mechanical stress, voltage, small molecules including capsaicin and menthol, and lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂). Since the same TRP channels may respond to different physical and chemical stimuli, they can serve as signal integrators. Many TRP channels are calcium permeable and contribute to Ca²⁺ homeostasis and signaling. Although the TRP channel family was discovered decades ago, only recently have the structures of many of these channels been solved, largely by cryo-electron microscopy (cryo-EM). Complimentary to cryo-EM, X-ray crystallography provides unique tools to unambiguously identify specific atoms and can be used to study ion binding in channel pores. In this review we describe crystallographic studies of the TRP channel TRPV6. The methodology used in these studies may serve as a template for future structural analyses of different types of TRP and other ion channels.     

Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography

Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid. We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model. The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds. The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.     

Combining X-ray crystallography and electron microscopy

The combination of cryo-electron microscopy to study large biological assemblies at low resolution with crystallography to determine near atomic structures of assembly fragments is quickly expanding the horizon of structural biology. This technique can be used to advantage in the study of large structures that cannot be crystallized, to follow dynamic processes, and to "purify" samples by visual selection of particles. Factors affecting the quality of cryo-electron microscopy maps and limits of accuracy in fitting known structural fragments are discussed.     

The structure of DNA as deduced from fiber X-ray crystallography

X-ray diffraction patterns obtained experimentally for fibers, together with their chemical structures, can be analyzed theoretically in terms of an integral equation. The partially unknown electron density function can be solved by iteration. This mathematical technique has been applied with success to study the secondary structures of DNA fibers.     

Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.     

A new drug binding subsite on human serum albumin and drug-drug interaction studied by X-ray crystallography

3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.     

Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering

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XRayView: a teaching aid for X-ray crystallography.

A software package, XRayView, has been developed that uses interactive computer graphics to introduce basic concepts of x-ray diffraction by crystals, including the reciprocal lattice, the Ewald sphere construction, Laue cones, the wavelength dependence of the reciprocal lattice, primitive and centered lattices and systematic extinctions, rotation photography. Laue photography, space group determination and Laue group symmetry, and the alignment of crystals by examination of reciprocal space. XRayView is designed with "user-friendliness" in mind, using pull-down menus to control the program. Many of the experiences of using real x-ray diffraction equipment to examine crystalline diffraction can be simulated. Exercises are available on-line to guide the users through many typical x-ray diffraction experiments.     

Structural insights into binding of inhibitors to soluble epoxide hydrolase gained by fragment screening and X-ray crystallography

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein–ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH.     

Fragment-based screening using X-ray crystallography and NMR spectroscopy

Approaches which start from a study of the interaction of very simple molecules (fragments) with the protein target are proving to be valuable additions to drug design. Fragment-based screening allows the complementarity between a protein active site and drug-like molecules to be rapidly and effectively explored, using structural methods. Recent improvements in the intensities of laboratory X-ray sources permits the collection of greater amounts of high-quality diffraction data and have been matched by developments in automation, crystallisation and data analysis. Developments in NMR screening, including the use of cryogenically cooled NMR probes and (19)F-containing reporter molecules have expanded the scope of this technique, while increasing the availability of binding site and quantitative affinity data for the fragments. Application of these methods has led to a greater knowledge of the chemical variety, structural features and energetics of protein-fragment interactions. While fragment-based screening has already been shown to reduce the timescales of the drug discovery process, a more detailed characterisation of fragment screening hits can reveal unexpected similarities between fragment chemotypes and protein active sites leading to improved understanding of the pharmacophores and the re-use of this information against other protein targets.     

Protein dynamics from X-ray crystallography: anisotropic, global motion in diffuse scattering patterns

Understanding X-ray crystallographic diffuse scattering is likely to improve our comprehension of equilibrium collective protein dynamics. Here, using molecular dynamics (MD) simulation, a detailed analysis is performed of the origins of diffuse scattering in crystalline Staphylococcal nuclease, for which the complete diffuse scattering pattern has been determined experimentally. The hydrogen-atom contribution and the scattering range over which the scattering can be considered to be a sum of solvent and protein scattering are determined. Two models of correlated protein motion are investigated by calculating the model-derived diffuse scattering and comparing with the scattering calculated directly from MD trajectories. In one model, previously used in diffuse scattering interpretation, the atomic displacement correlations decay isotropically with increasing separation. Model correlation lengths are obtained by refining the model scattering against the simulation-derived scattering pattern, and are found to be significantly different from those correlation lengths derived directly from the MD trajectories. Furthermore, the convergence between the model-derived and MD-derived scattering is poor. The second model, in which the displacement correlations are calculated from the principal components of the MD trajectories, is capable of fully reproducing the MD-derived diffuse scattering if the approximately 50% lowest-frequency modes are included. However, a small number ( approximately 10) of lowest-frequency and largest-amplitude modes dominates the diffuse scattering and thus the correlated protein motions. A detailed analysis of the principal components is performed. In particular, the effective free energy profile associated with each principle mode is analyzed and the eigenfrequency and damping coefficient computed using a model of Brownian dynamics. Those collective modes with effective frequencies below approximately 0.5 THz, including those that determine the diffuse scattering, are overdamped.