High-resolution Structural Analysis Research Articles

Interaction studies of Alzheimer's Cathepsin B protein with inhibitors in presence and absence of water

Background The accuracy of ligand-protein docking may be affected by the presence of water molecules on the surface of proteins. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A recent analysis of high-resolution crystal structures of ligand-protein complexes revealed that 85% of the complexes had one or more water molecules bridging the interaction between ligand and protein. For predicting the binding modes and energies of protein-ligand interactions, molecular docking methods are commonly used. In order to obtain an accurate complex geometry and binding energy estimation, an appropriate method for calculating partial charges is essential. AutoDockTools software, widely used as interface for preparing input files, utilizes the either Gasteiger or Kollman partial charge calculation method for both protein and ligand charge calculations. However, it has already been reported that more accurate partial charge calculation and as a consequence, more accurate docking can be achieved by using quantum chemical methods. In common practice so far, the quantum chemical partial charges were applied to the ligands for docking calculations. The newly developed Mozyme function of MOPAC2009 allows fast partial charge calculation of proteins by quantum mechanical semiempirical methods. Thus, in the current study, we use the semi-empirical quantum-mechanical partial charge calculations to investigate the interaction energies and polarization effects of the various components of the binding pocket on a set of Cathepsin B protein. Results The docking accuracy was computed by using the original AutoDock scoring function with the set of 19 protein ligand complexes using Gasteiger, AM1 and PM3 partial charge calculation methods. This helped us to compare the effect of the partial charge calculation method on docking accuracy. It was seen that the docking accuracy in regard to complex geometry significantly increased when partial charges of the ligands and proteins were calculated with the semi-empirical PM3 method. Our results demonstrate that (i) the energetic of the key water molecule are more favorable for the binding site in the Cathepsin B protein (ii) Water bridging and triangle formation were seen between the key amino acid residue and the ligand (iii) The internal energy is significant factor for the binding modes of various ligands. It was also observed a statistically significant overall increase in accuracy when water molecules are included during docking simulations. Out of the 19 complexes analyzed in the course of our study, the geometry of 17 complexes were accurately calculated using PM3 partial charges, while the use of Gasteiger charges resulted in only 8 accurate geometries. Conclusion Our findings indicate that the inclusion of water molecules in ligand-protein docking results in significant increases in docking accuracy when the use of quantum chemical partial charge assignment on both ligand and proteins for predicting the docking simulations.

1P112 アジ化物結合型チトクロム酸化酵素の高分解能X線結晶構造解析によるアジ化物結合様式の解析(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

1P112 アジ化物結合型チトクロム酸化酵素の高分解能X線結晶構造解析によるアジ化物結合様式の解析(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

Breakthrough of cryo-TEM for high resolution structural analysis of protein complexes(The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Breakthrough of cryo-TEM for high resolution structural analysis of protein complexes(The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Protection against Lethal Enterovirus 71 Challenge in Mice by a Recombinant Vaccine Candidate Containing a Broadly Cross-Neutralizing Epitope within the VP2 EF Loop

Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the “puff” region that forms the “southern rim” of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.

Interfacial energies and mass transport in the Ni(Al)–Al2O3 system: The implication of very low oxygen activities

Adhesion and capillary-driven mass transport at ceramic–metal interfaces play a very important role in the performance and durability of materials for many applications, and the influence of the oxygen activity is a critical issue. This work systematically investigates the variation of interfacial energies and atomic transport mechanisms at metal–oxide interfaces at very low oxygen activities by bonding Ni–Al alloys and pure polycrystalline alumina under controlled conditions in sessile drop experiments. The angles and the evolution of the grain boundary grooves were analyzed by scanning electron microscopy, atomic force microscopy and focused ion beam milling to calculate the interfacial and grain boundary energies and the transport rates at the metal–Al2O3 interface. In parallel, high-resolution structural and chemical analysis of selected grain boundaries was performed using advanced transmission electron microscopy. Our results confirm that all the interfacial energies (metal–Al2O3, Al2O3 surface and grain boundary energy) are smaller at reduced p(O2) than those of stoichiometric interfaces. The atomic transport at the metal–Al2O3 interface was found to decrease initially with decreasing p(O2) but increased significantly with a further decrease in the oxygen activity.

Imperfect coordination chemistry facilitates metal ion release in the Psa permease

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.

Synthesis and Microdiffraction at Extreme Pressures and Temperatures

High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary interiors, design materials with novel properties, understand the mechanical behavior of materials exposed to very high stresses as in explosions or impacts. Synthesis and structural analysis of materials at extreme conditions of pressure and temperature entails remarkable technical challenges. In the laser heated diamond anvil cell (LH-DAC), very high pressure is generated between the tips of two opposing diamond anvils forced against each other; focused infrared laser beams, shined through the diamonds, allow to reach very high temperatures on samples absorbing the laser radiation. When the LH-DAC is installed in a synchrotron beamline that provides extremely brilliant x-ray radiation, the structure of materials under extreme conditions can be probed in situ. LH-DAC samples, although very small, can show highly variable grain size, phase and chemical composition. In order to obtain the high resolution structural analysis and the most comprehensive characterization of a sample, we collect diffraction data in 2D grids and combine powder, single crystal and multigrain diffraction techniques. Representative results obtained in the synthesis of a new iron oxide, Fe4O5 (1) will be shown.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design

Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state(1). However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging(2), due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-1(3-7), the resolution afforded by live-cell microscopy is limited (~200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target. In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

Large-Scale Synthesis of Bicrystalline ZnO Nanowire Arrays by Thermal Oxidation of Zinc Film: Growth Mechanism and High-Performance Field Emission

Understanding the origin crystal nucleation and driving force is critical for the synthesis of one-dimensional nanomaterials with controllable size, morphology, and crystal structure. The growth behavior of ZnO nanowires prepared by thermal oxidation of zinc film is studied. It is found that the grown nanowires have a bicrystalline structure and growth direction significantly different from the commonly observed [0001] direction. On the basis of detailed high resolution morphology and structural analysis, we propose the origin of the initial growth site, as well as the driving force for formation of the bicrystalline structure of aligned ZnO nanowires. The initial zinc film plays an important role in the growth of nanowire. The edge-enhanced oxidation effect of zinc grain initiates the ZnO nanowire nucleation. The strain within the ZnO layer drives and stimulates the nanowire growth. The present study provides insight into the growth mechanism of ZnO nanowires grown from thermal oxidation of zinc film. Field emission measurement results show that the prepared ZnO nanowires have excellent field emission properties. Uniform emission can be obtained and the turn-on field is 7.8 V/μm. The results indicate that the method is advantageous for large-scale synthesis of ZnO nanowires for field emission applications.

Increasing the electron-transfer ability of Cyanidioschyzon merolae ferredoxin by a one-point mutation – A high resolution and Fe-SAD phasing crystal structure analysis of the Asp58Asn mutant

Cyanidioschyzon merolae (Cm) is a single cell red algae that grows in rather thermophilic (40–50°C) and acidic (pH 1–3) conditions. Ferredoxin (Fd) was purified from this algae and characterized as a plant-type [2Fe–2S] Fd by physicochemical techniques. A high resolution (0.97Å) three-dimensional structure of the CmFd D58N mutant molecule has been determined using the Fe-SAD phasing method to clarify the precise position of the Asn58 amide, as this substitution increases the electron-transfer ability relative to wild-type CmFd by a factor of 1.5. The crystal structure reveals an electro-positive surface surrounding Asn58 that may interact with ferredoxin NADP+ reductase or cytochrome c.

How oxidized grain boundaries fail

Macroscopic behaviour of materials is often controlled by microscopic events; this has driven interest in testing and analysing increasingly smaller features. The ability to perform mechanical tests on the micron scale, with modelling and high-resolution chemical and structural analysis on the same scale, now makes it possible to investigate in detail the mechanisms controlling one of the most complex modes of fracture: stress corrosion cracking (SCC). In this paper, through such a multifaceted approach, we show that individual grain boundaries, preferentially oxidized after exposure to simulated pressurized nuclear reactor cooling water, can be mechanically tested and their resistance to fracture quantified. These results have direct consequences in understanding the mechanisms controlling SCC propagation and initiation.

Protein production from the structural genomics perspective: achievements and future needs.

Despite a multitude of recent technical breakthroughs speeding high-resolution structural analysis of biological macromolecules, production of sufficient quantities of well-behaved, active protein continues to represent the rate-limiting step in many structure determination efforts. These challenges are only amplified when considered in the context of ongoing structural genomics efforts, which are now contending with multi-domain eukaryotic proteins, secreted proteins, and ever-larger macromolecular assemblies. Exciting new developments in eukaryotic expression platforms, including insect and mammalian-based systems, promise enhanced opportunities for structural approaches to some of the most important biological problems. Development and implementation of automated eukaryotic expression techniques promises to significantly improve production of materials for structural, functional, and biomedical research applications.

Molecular signatures of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

Open questions: Chromosome condensation - Why does a chromosome look like a chromosome?

The x-shaped appearance of a chromosome in mitosis has become one of the iconic symbols of the life sciences (Figure ​(Figure1).1). DNA takes its chromosome shape only during cell division, but for that time, the compaction of the genomic DNA is critical. The human genome consists of several meters of DNA: even divided between 46 chromosomes, this still leaves several centimeters to be packed into each chromosome. One might be tempted to invoke a magician's hand at work to package centimeters of DNA into a micrometer-sized chromosome. But of course, there is no magic in biology. Nor is there, yet, an answer to the question of how it is done. Figure 1 Human chromosomes. Meters of DNA are packed into micrometer-sized chromosomes in mitosis, here in human (HeLa) cells arrested in mitosis. Chromosomes were spread and stained with a DNA binding dye. Scale bar, 10 μm (photograph courtesy of Sriramkumar ... Our current understanding of chromosome architecture at the molecular level is still far from complete. The only thing that becomes increasingly clear is that long-standing textbook models that show a hierarchical folding of DNA into coils and loops of increasing diameter [1], that eventually make up chromosome arms, is wrong. But if not in hierarchical coils, how is DNA folded inside a mitotic chromosome? Much of the DNA is of course incorporated into nucleosomes, but apart from that little is known about the path of DNA inside a chromosome. New genomic techniques coupled to the astounding present-day DNA sequencing power [2] and high-resolution structural analyses using a combination of modern methods [3] are beginning to give us a new sense of chromosome organization. The sense is of an irregular, flexible and self-organizing system without a predetermined folding pattern. Thus, to appreciate the behavior of a nucleosome fiber in a chromosome we shall have to consider its biophysics. Molecular dynamics simulations will be key to understanding the implications of the biophysical constraints at a mesoscale level. Ultimately, it will be proteins and protein complexes that organize the DNA in a mitotic chromosome. Linker histones will play a role in organisms that encode them; but the density of DNA in a human chromosome is not very different from that in a bacterial nucleoid, which does not have nucleosomes. The packaging proteins that are universal are the members of the structural maintenance of chromosomes (SMC) family, which have been implicated in chromosome compaction in vertebrates and in bacteria for a long time [4-6]. How these and other proteins work on chromosomes, and indeed make a chromosome look like a chromosome, are questions that will hopefully not remain open for much longer.

The impulsivity behavior is correlated with prefrontal cortex gray matter volume reduction in heroin-dependent individuals

PurposeChronic exposure to heroin induced cerebral structural abnormalities may underlie heroin-related behaviors. The aim of this study was to: (1) identify cerebral structural abnormalities in heroin-dependent individuals (HDIs) by an automated and unbiased morphometric technique. (2) Define the correlation between these cerebral structural abnormalities and the impulsivity characteristic in HDIs. Methods24 HDIs and 24 control subjects were completed with (1) high resolution structural magnetic resonance imaging scanning and analysis of gray matter volume using voxel-based morphometry implemented in Statistical Parametric Mapping and (2) a Chinese translation Barratt Impulsiveness Scale-11 questionnaire survey. Differences in regional gray matter volume were tested using an analysis of covariance model, co-varying for global gray matter and age. Statistical maps were set at p<0.05, corrected for multiple comparisons. The abnormal brain regions were correlated with the duration of heroin use and impulsivity scores. ResultsAfter adjusting for effects of age and total gray matter volume, cortical gray matter volume in the bilateral medial prefrontal cortex, bilateral dorsal lateral prefrontal cortex, and right fusiform cortex were significantly reduced in HDIs. Moreover, the gray matter volume in prefrontal cortex that showed group differences was negatively correlated with the duration of heroin use and negatively correlated with the impulsivity characteristic in HDIs. ConclusionThese findings reveal the prefrontal cortex was impaired in HDIs, meanwhile, indicate the changes in gray matter volume are relating to the duration of heroin use and the impulsivity characteristic of the HDIs.

CENP-T provides a structural platform for outer kinetochore assembly

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner.

Structure, function and biosynthesis of O2-tolerant hydrogenases

Molecular hydrogen (H(2)) is used as an energy source or a way to deposit excess reducing power by a wide range of microorganisms. Both H(2) oxidation and production are catalysed by hydrogenases. As these metalloenzymes are usually exquisitely O(2) sensitive, H(2) metabolism under aerobic conditions, which is known as O(2)-tolerant H(2) cycling, involves hydrogenases that have undergone structural and catalytic adaptations and requires a dedicated biosynthetic machinery. Here, we discuss recent high-resolution crystal structure analyses of a particular subtype of [NiFe]-hydrogenase that is predominantly found in aerobic or facultative aerobic H(2)-oxidizing bacteria. These data have provided insights into the underlying molecular strategies that allow sustained biological conversion of H(2) in the presence of O(2).

1P084 高分解能構造解析に向けた電子顕微鏡用カメラの評価(01E. 蛋白質:計測・解析の方法論,ポスター,日本生物物理学会年会第51回(2013年度))

1P084 高分解能構造解析に向けた電子顕微鏡用カメラの評価(01E. 蛋白質:計測・解析の方法論,ポスター,日本生物物理学会年会第51回(2013年度))

Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images

Data mining from noisy data/images is one of the most important themes in modern science and technology. Statistical image processing is a promising technique for analysing such data. Automation of particle pickup from noisy electron micrographs is essential, especially when improvement of the resolution of single particle analysis requires a huge number of particle images. For such a purpose, reference-based matching using primary three-dimensional (3D) model projections is mainly adopted. In the matching, however, the highest peaks of the correlation may not accurately indicate particles when the image is very noisy. In contrast, the density and the heights of the peaks should reflect the probability distribution of the particles. To statistically determine the particle positions from the peak distributions, we have developed a density-based peak search followed by a peak selection based on average peak height, using multi-reference alignment (MRA). Its extension, using multi-reference multiple alignment (MRMA), was found to enable particle pickup at higher accuracy even from extremely noisy images with a signal-to-noise ratio of 0.001. We refer to these new methods as stochastic pickup with MRA (MRA-StoPICK) or with MRMA (MRMA-StoPICK). MRMA-StoPICK has a higher pickup accuracy and furthermore, is almost independent of parameter settings. They were successfully applied to cryo-electron micrographs of Rice dwarf virus. Because current computational resources and parallel data processing environments allow somewhat CPU-intensive MRA-StoPICK and MRMA-StoPICK to be performed in a short period, these methods are expected to allow high-resolution analysis of the 3D structure of particles.