Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Zuanning Yuan , Bo Yin , Dongzhi Wei , Yu-ren Adam Yuan

Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Cyanobacterium Synechococcus contains one ALDH enzyme (Sp2771), together with a novel 2-oxoglutarate decarboxylase, to complete a non-canonical tricarboxylic acid cycle. However, the molecular mechanisms for substrate selection and cofactor preference by Sp2771 are largely unknown. Here, we report crystal structures of wild type Sp2771, Sp2771 S419A mutant and ternary structure of Sp2771 C262A mutant in complex with NADP+ and SSA, as well as binary structure of Gluconobacter oxydans aldehyde dehydrogenase (Gox0499) in complex with PEG. Structural comparison of Sp2771 with Gox0499, coupled with mutational analysis, demonstrates that Ser157 residue in Sp2771 and corresponding Pro159 residue in Gox0499 play critical structural roles in determining NADP+ and NAD+ preference for Sp2771 and Gox0499, respectively, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. Hence, our work has provided insightful structural information into cofactor and substrate selection by ALDH.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Sébastien Moniot , Mike Schutkowski , Clemens Steegborn

Sirtuins are NAD+-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Tri-Nhan Nguyen , Dong-Ju You , Hiroyuki Matsumoto , Eiko Kanaya , Yuichi Koga , Shigenori Kanaya

LC11-RNase H1 is a Sulfolobus tokodaii RNase H1 (Sto-RNase H1) homologue isolated by metagenomic approach. In this study, the crystal structure of LC11-RNase H1 in complex with an RNA/DNA substrate was determined. Unlike Bacillus halodurans RNase H1 without hybrid binding domain (HBD) (Bh-RNase HC) and human RNase H1 without HBD (Hs-RNase HC), LC11-RNase H1 interacts with four non-consecutive 2′-OH groups of the RNA strand. The lack of interactions with four consecutive 2′-OH groups leads to a dramatic decrease in the ability of LC11-RNase H1 to cleave the DNA–RNA–DNA/DNA substrate containing four ribonucleotides as compared to those to cleave the substrates containing five and six ribonucleotides. The interaction of LC11-RNase H1 with the DNA strand is also different from those of Bh-RNase HC and Hs-RNase HC. Beside the common phosphate-binding pocket, LC11-RNase H1 has a unique DNA-binding channel. Furthermore, the active-site residues of LC11-RNase H1 are located farther away from the scissile phosphate group than those of Bh-RNase HC and Hs-RNase HC. Modeling of Sto-RNase H1 in complex with the 14bp RNA/DNA substrate, together with the structure-based mutational analyses, suggest that the ability of Sto-RNase H1 to cleave double-stranded RNA is dependent on the local conformation of the basic residues located at the DNA binding site.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Craig Yoshioka , Dmitry Lyumkis , Bridget Carragher , Clinton S. Potter

Electron microscopy (EM) is an important tool for determining the composition, arrangement and structure of biological macromolecules. When studying structurally heterogeneous samples using EM, classification is a critical step toward achieving higher resolution and identifying biologically significant conformations. We have developed an interactive, web-based tool, called Maskiton, for creating custom masks and performing 2D classifications on aligned single-particle EM images. The Maskiton interface makes it considerably easier and faster to explore the significance of heterogeneity in single-particle datasets. Maskiton features include: resumable uploads to facilitate transfer of large datasets to the server, custom mask creation in the browser, continual progress updates, and interactive viewing of classification results. To demonstrate the value of this tool, we provide examples of its use on several experimental datasets and include analyses of the independent terminus mobility within the Ltn1 E3 ubiquitin ligase, the in vitro assembly of 30S ribosomal subunits, and classification complexity reduction within Immunoglobulin M. This work also serves as a proof-of-concept for the development of future cross-platform, interactive user interfaces for electron microscopy data processing.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Beata Jastrzebska , Philippe Ringler , Krzysztof Palczewski , Andreas Engel

Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho∗, which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-β-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho∗·Gt and sConA-Rho∗·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho∗·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho∗·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho∗·Gt complex in which the photoactivated Rho∗ dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho∗ dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Andreas D. Schenk , Ansgar Philippsen , Andreas Engel , Thomas Walz

Electron crystallography of two-dimensional crystals allows the structural study of membrane proteins in their native environment, the lipid bilayer. Determining the structure of a membrane protein at near-atomic resolution by electron crystallography remains, however, a very labor-intense and time-consuming task. To simplify and accelerate the data processing aspect of electron crystallography, we implemented a pipeline for the processing of electron diffraction data using the Image Processing Library and Toolbox (IPLT), which provides a modular, flexible, integrated, and extendable cross-platform, open-source framework for image processing. The diffraction data processing pipeline is organized as several independent modules implemented in Python. The modules can be accessed either from a graphical user interface or through a command line interface, thus meeting the needs of both novice and expert users. The low-level image processing algorithms are implemented in C++ to achieve optimal processing performance, and their interface is exported to Python using a wrapper. For enhanced performance, the Python processing modules are complemented with a central data managing facility that provides a caching infrastructure. The validity of our data processing algorithms was verified by processing a set of aquaporin-0 diffraction patterns with the IPLT pipeline and comparing the resulting merged data set with that obtained by processing the same diffraction patterns with the classical set of MRC programs.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Konstantin V. Korotkov , Wim G.J. Hol

Bacteria contain several sophisticated macromolecular machineries responsible for translocating proteins across the cell envelope. One prominent example is the type II secretion system (T2SS), which contains a large outer membrane channel, called the secretin. These gated channels require specialized proteins, so-called pilotins, to reach and assemble in the outer membrane. Here we report the crystal structure of the pilotin GspS from the T2SS of enterohemorrhagic Escherichia coli (EHEC), an important pathogen that can cause severe disease in cases of food poisoning. In this four-helix protein, the straight helix α2, the curved helix α3 and the bent helix α4 surround the central N-terminal helix α1. The helices of GspS create a prominent groove, mainly formed by side chains of helices α1, α2 and α3. In the EHEC GspS structure this groove is occupied by extra electron density which is reminiscent of an α-helix and corresponds well with a binding site observed in a homologous pilotin. The residues forming the groove are well conserved among homologs, pointing to a key role of this groove in this class of T2SS pilotins. At the same time, T2SS pilotins in different species can be entirely different in structure, and the pilotins for secretins in non-T2SS machineries have yet again unrelated folds, despite a common function. It is striking that a common complex function, such as targeting and assembling an outer membrane multimeric channel, can be performed by proteins with entirely different folds.

Publication date: May 2013
Source:Journal of Structural Biology, Volume 182, Issue 2

Author(s): Kazutaka Murayama , Kota Kano , Yusaku Matsumoto , Daisuke Sugimori

The metal-independent lipase from Streptomyces albidoflavus NA297 (SaPLA1) is a phospholipase A1 as it preferentially hydrolyzes the sn-1 acyl ester in glycerophospholipids, yielding a fatty acid and 2-acyl-lysophospholipid. The molecular mechanism underlying the substrate binding by SaPLA1 is currently unknown. In this study, the crystal structure of SaPLA1 was determined at 1.75Å resolutions by molecular replacement. A structural similarity search indicated the highest structural similarity to an esterase from Streptomyces scabies, followed by GDSL family enzymes. The SaPLA1 active site is composed of a Ser-His dyad (Ser11 and His218), whereby stabilization of the imidazole is provided by the main-chain carbonyl oxygen of Ser216, a common variation of the catalytic triad in many serine hydrolases, where this carbonyl maintains the orientation of the active site histidine residue. The hydrophobic pocket and cleft for lipid binding are adjacent to the active site, and are approximately 13–15Å deep and 14–16Å long. A partial polyethylene glycol structure was found in this hydrophobic pocket.

Publication date: Available online 25 April 2013
Source:Journal of Structural Biology

Author(s): Paul H. Backe , Roger Simm , Jon K. Laerdahl , Bjørn Dalhus , Annette Fagerlund , Ole A. Økstad , Torbjørn Rognes , Ingrun Alseth , Anne-Brit Kolstø , Magnar Bjørås

The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique β-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.

Publication date: Available online 18 April 2013
Source:Journal of Structural Biology

Author(s): Wei Zhang , Bärbel Kaufmann , Paul R. Chipman , Richard J. Kuhn , Michael G. Rossmann

Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by icosahedral scaffolding composed of envelope (E) and membrane (M) proteins through interaction of the proteins’ stem-anchor regions with the membrane. In localized regions, small membrane regions exhibit convex, concave, flat or saddle-shaped surfaces that are constrained by the specific protein organization within each membrane leaflet. These results suggest that the organization of membrane proteins in small enveloped viruses mediate the formation of membrane curvature.

Publication date: Available online 18 April 2013
Source:Journal of Structural Biology

Author(s): Avanish S. Parmar , Sohail Zahid , Sandeep Belure , Robert Young , Nida Hasan , Vikas Nanda

Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances specificity of target state on the expense of its stability.

Publication date: Available online 17 April 2013
Source:Journal of Structural Biology

Author(s): Sabrina Reich , Nico Kress , Bettina M. Nestl , Bernhard Hauer

The engineering of protein stability is of major importance for the application of enzymes in a wide range of industrial applications. Here we study the determinants of the thermo- and solvent stability of the Zymomonas mobilis ene reductase NCR using a rational protein engineering approach based on analyses of structural and sequence data. We designed and created two loop mutants with the aim to increase their overall stability. They all retained catalytic activity but exhibited altered thermostability relative to the wild-type enzyme. The modulation of one specific loop segment near the active site of NCR showed an increased tolerance to organic solvents along with an enhanced thermostability.

Publication date: Available online 15 April 2013
Source:Journal of Structural Biology

Author(s): Fabio Nudelman , Alexander J. Lausch , Nico A.J.M. Sommerdijk , Eli D. Sone

Over the last several years, significant progress has been made toward understanding the mechanisms involved in the mineralization of hard collagenous tissues, such as bone and dentin. Particularly notable are the identification of transient mineral phases that are precursors to carbonated hydroxyapatite, the identification and characterization of non-collagenous proteins that are involved in controlling mineralization, and significant improvements in our understanding of the structure of collagen. These advances not only represent a paradigm shift in the way collagen mineralization is viewed and understood, but have also brought new challenges to light. In this review, we discuss how recent in vitro models have addressed critical questions regarding the role of the non-collagenous proteins in controlling mineralization, the nature of the interactions between amorphous calcium phosphate and collagen during the early stages of mineralization, and the role of collagen in the mineralization process. We discuss the significance of these findings in expanding our understanding of collagen biomineralization, while addressing some of the limitations that are inherent to in vitro systems.

Publication date: Available online 11 April 2013
Source:Journal of Structural Biology

Author(s): Fred Wilt , Christopher E. Killian , Lindsay Croker , Patricia Hamilton

A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO’s) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.

Publication date: Available online 10 April 2013
Source:Journal of Structural Biology

Author(s): Felipe Eltit , Vincent Ebacher , Rizhi Wang

Dentin is a mineralized collagen tissue with robust mechanical performance. Understanding the mechanical behavior of dentin and its relations to the dentinal structure can provide insight into the design strategies to achieve tooth functions. This study focuses on the inelastic deformation of human dentin and its underlying mechanisms. By combining four-point bending tests with fluorescent staining and laser scanning confocal microscopy, it was found that human dentin, especially root dentin, exhibited significant inelastic deformation and developed extensive microdamage in the form of microcracks prior to fracture. Dense and wavy microcracks spread uniformly across the tensile surface of root dentin, while compressive microcracks formed cross-hatched patterns. The presence of peritubular dentin in coronal dentin dramatically decreased the extent of microcracking, reducing inelasticity. Dentinal tubules were found to be initiation sites of both tensile and compressive microcracks. A unique crack propagation process was observed in root dentin under tension: numerous ring-shaped cracks formed at each dentinal tubule ahead of a growing crack tip. The advance of the tensile microcracks occurred by the merging of those ring-shaped cracks. The current findings on the microcracking process associated with inelastic deformation helps to understand the nature of strength and toughness in dentin, as well as the mechanical significance for structural variations across the whole tooth.

Publication date: Available online 6 April 2013
Source:Journal of Structural Biology

Author(s): Claudia Antoni , Laura Vera , Laurent Devel , Maria Pia Catalani , Bertrand Czarny , Evelyn Cassar-Lajeunesse , Elisa Nuti , Armando Rossello , Vincent Dive , Enrico Adriano Stura

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.

Publication date: Available online 4 April 2013
Source:Journal of Structural Biology

Author(s): Victoria Gallon , Lisha Chen , Xiudong Yang , Janet Moradian-Oldak

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1–8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45μm height, 33μm width) and small (7μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.

Publication date: Available online 4 April 2013
Source:Journal of Structural Biology

Author(s): Cyril Hamiaux , Lisa Basten , David R. Greenwood , Edward N. Baker , Richard D. Newcomb

Takeout proteins are found across a diverse range of insect species and are thought to be involved in important aspects of insect physiology and behavior. These proteins act as ligand carriers, but the nature of their endogenous ligands remains unknown. The crystal structure of Epiphyas postvittana Takeout 1 (EpTo1), the only structure for any Takeout protein to date, revealed an α/β-wrap fold with a purely hydrophobic internal cavity of tubular shape. When recombinantly expressed in Escherichia coli, we previously showed that a surrogate ubiquinone-8 ligand binds within the internal cavity of EpTo1 with excellent shape complementarity. We have now expressed EpTo1 in an insect cell expression system devoid of ubiquinone-8, and solved its crystal structure at 2.2Å resolution. Using combined information from crystallography and mass spectrometry, we identify a mixture of fatty acid moieties, mostly myristic and palmitic acid, bound inside the EpTo1 cavity, mimicking the structure of the longer ubiquinone-8 compound. No significant alteration of the internal cavity was observed regardless of the bound ligands, ubiquinone-8 or fatty acids, suggesting that the internal cavity of EpTo1 forms a rigid scaffold that imposes strict structural constraints for selectivity and specificity of ligand(s) in vivo.

Publication date: April 2013
Source:Journal of Structural Biology, Volume 182, Issue 1

Publication date: April 2013
Source:Journal of Structural Biology, Volume 182, Issue 1