High-resolution Protein Crystallography Research Articles

Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-β-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a − 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.

Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

Nickel-Iron Hydrogenases: High-Resolution Crystallography Resolves the Hydride, but Not the Debate.

An (X-ray) eye for detail: Modern high-resolution protein crystallography allows H atoms to be located. Applied to nickel-iron hydrogenase, X-ray structural analysis has finally confirmed the presence of an active-site hydride and thiol, as well as unveiling the intricate pathways that protons take to and from the active site.

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV–visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

構造生物学研究の新展開 １．タンパク質構造研究に対する結晶学の役割 タンパク質における超高分解能Ｘ線結晶構造解析

Many protein structures have been determined by X-ray crystallography and deposited with the Protein Data Bank. However, these structures at usual resolution (1.5<d<3.0 Å) are insufficient in their precision and quantity for elucidating the molecular mechanism of protein functions directly from structural information. Several studies at ultra-high resolution (d<0.8 Å) have been performed with synchrotron radiation in the last decade. The highest resolution of the protein crystals was achieved at 0.54 Å resolution for a small protein, crambin. In such high resolution crystals, almost all of hydrogen atoms of proteins and some hydrogen atoms of bound water molecules are experimentally observed. In addition, outer-shell electrons of proteins can be analyzed by the multipole refinement procedure. However, the influence of X-rays should be precisely estimated in order to derive meaningful information from the crystallographic results. In this review, we summarize refinement procedures, current status and perspectives for ultra high resolution protein crystallography.

Structure determinations of human and chimaeric antibodies by solution scattering and constrained molecular modelling

X-ray and neutron scattering and analytical ultracentrifugation provide multiparameter structural and compositional information on proteins that complements high-resolution protein crystallography and NMR studies. They are ideal methods to use when either a large protein cannot be crystallized, when scattering provides the only means to obtain a solution structure, or the protein crystal structure has been determined and it is necessary to validate this. Once these results have been obtained, we apply automated constrained modelling methods based on known subunit crystal structures to identify the best-fit structure. Using our antibody structures as examples, we describe the generation of appropriate starting models, randomizing these for trial-and-error scattering fits, identifying the final best-fit models and interpreting these in terms of function. We discuss our structure determinations for IgA and IgD, an IgA-human serum albumin complex, the dimer of IgA and secretory component associated with this and chimaeras of mouse IgG with two complement proteins. Constrained modelling confirms the experimental data analysis and produces families of best-fit molecular models. Its usage has clarified several aspects of antibody structure and function in solution.

Anisotropic atomic motions in high-resolution protein crystallography molecular dynamics simulations

Molecular dynamics (MD) simulations using empirical force fields are popular for the study of proteins. In this work, we compare anisotropic atomic fluctuations in nanosecond-timescale MD simulations with those observed in an ultra-high-resolution crystal structure of crambin. In order to make our comparisons, we have developed a compact graphical technique for assessing agreement between spatial atomic distributions determined by MD simulations and observed anisotropic temperature factors.

Getting protein solvent structures down cold

Getting protein solvent structures down cold

Water-protein interactions from high-resolution protein crystallography.

To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level.

The PDZ2 Domain of Syntenin at Ultra-high Resolution: Bridging the Gap Between Macromolecular and Small Molecule Crystallography

The crystal structure of the second PDZ domain of the scaffolding protein syntenin was solved using data extending to 0.73 Å resolution. The crystallographic model, including the hydrogen atoms and the anisotropic displacement parameters, was refined to a conventional R-factor of 7.5% and R free of 8.7%, making it the most precise crystallographic model of a protein molecule to date. The model reveals discrete disorder in several places in the molecule, and significant plasticity of the peptide bond, with some ω angles deviating by nearly 20° from planarity. Most hydrogen atoms are easily identifiable in the electron density and weak hydrogen bonds of the C–H⋯O type are clearly visible between the β-strands. The study sets a new standard for high-resolution protein crystallography.

The ‘glass transition’ in protein dynamics: what it is, why it occurs, and how to exploit it

All proteins undergo a dramatic change in their dynamical properties at approximately 200 K. Above this temperature, their dynamic behavior is dominated by large-scale collective motions of bonded and nonbonded groups of atoms. At lower temperatures, simple harmonic vibrations predominate. The transition has been described as a ‘glass transition’ to emphasize certain similarities between the change in dynamic behavior of individual protein molecules and the changes in viscosity and other properties of liquids when they form a glass. The glass transition may reflect the intrinsic temperature dependence of the motions of atoms in the protein itself, in the bound solvent on the surface of the protein, or it may reflect contributions from both. Protein function is significantly altered below this transition temperature; a fact that can be exploited to trap normally unstable intermediates in enzyme-catalyzed reactions and stabilize them for periods long enough to permit their characterization by high-resolution protein crystallography.

Performance and applications of a high rate imaging pixel detector

In the past years a large variety of gas filled micro pattern detectors have been developed for applications in high energy physics as well as for X-ray imaging in synchrotron light experiments. Here, we present the most recent developments on the MicroCAT detector with resistive position encodeing readout, which has been demonstrated to meet even the strong requirements in high resolution protein crystallography and time resolved small angle scattering at synchrotron light sources of the third generation. Recent test measurements with the prototype under the working conditions of a synchrotron light source as well as high time resolved measurements in the laboratory are presented.

Predictions of protein backbone bond distances and angles from first principles

Using φ(NC α) ψ(C αC′) conformational geometry maps of N-acetyl- N′-methylalanine amide obtained from ab initio calculations, a simple procedure has been developed that allows one to model the backbone bond distances and angles of proteins as functions of the φ and ψ torsional angles. For bond angles, comparisons with protein crystal structures can be made, and calculated conformational variations in bond distances (several hundredths of an Å) are more precise than the current resolution of experimental techniques. For the important backbone parameter NC αC′, the accuracy of the calculations is found to be comparable with that of high resolution protein crystallography. Further improvements are possible by applying empirical corrections. For example, the dipeptide derived modeled values of N C αC′ are consistently ≈ 2–3 ° above the values found in helical regions of oligopeptides and proteins. The procedure promises to be useful in many practical applications, for example in peptide and protein modeling and in the refinement of protein crystal data.

Cylindrical film cassette for high-resolution protein crystallography

Cylindrical film cassette for high-resolution protein crystallography

An oscillation data collection system for high-resolution protein crystallography

An oscillation data collection system for protein crystallography is described. The system consists of a modified Enraf-Nonius precession camera with cylindrical cassette, and stepping motor driven by a flexible microprocessor control system. The X-ray source is graphite-monochromatized radiation from an Elliott GX-21 rotating-anode generator run at 5.5 kW on a focal spot measuring 0.5 × 5.0 mm. The potential advantages of using a relatively large focal spot in conjunction with a graphite monochromator are discussed. Conditions for optimum collimation and X-ray intensity are considered, and it is shown that appropriately designed collimators with adjustable apertures can have substantial advantages over commercially available pinhole collimators. The oscillation films are processed by a procedure based on that of Rossmann [J. Appl. Cryst. (1979), 12, 225-238]. Determination of the initial alignment of the film is facilitated by a pair of reference pins incorporated in the cylindrical cassette. These pins ensure that the position of the film in the cassette is known, and avoid the need for fiducial marks. The crystal alignment and film measurement technique is fully automatic, requiring no prior input other than the approximate starting orientation of the crystal, the approximate unit-cell dimensions, and the angular oscillation range. An alternative method for the determination of crystal orientation is proposed which has been found to be somewhat superior to that of Rossmann, especially for smaller unit cells.