nils-herrmann/clean_scirepeval huggingface dataset:
Structural basis for pH dependent monomer-dimer transition of 3,4-dihydroxy 2-butanone-4-phosphate synthase domain from Mycobacterium tuberculosis.
Venue: Journal of structural biology

3,4-dihydroxy 2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase-II (GTPCH-II) are the two initial enzymes involved in riboflavin biosynthesis pathway, which has been shown to be essential for the pathogens. In Mycobacterium tuberculosis (Mtb), the ribA2 gene (Rv1415) encodes for the bi-functional enzyme with DHBPS and GTPCH-II domains at N- and C-termini, respectively. We have determined three crystal structures of Mtb-DHBPS domain in complex with phosphate and glycerol at pH 6.0, with sulphate at pH 4.0 and with zinc and sulphate at pH 4.0 at 1.8, 2.06 and 2.06 Å resolution, respectively. The hydrodynamic volume and enzyme activity studies revealed that the Mtb-DHBPS domain forms a functional homo-dimer between the pH 6.0 and 9.0, however, at pH 5.0 and below, it forms a stable inactive monomer in solution. Furthermore, the functional activity of Mtb-DHBPS and its dimeric state could be restored by increasing the pH between 6.0 and 9.0. The comparison of crystal structures determined at different pH revealed that the overall three-dimensional structure of Mtb-DHBPS monomer remains the same. However, the length of the α6-helix at pH 6.0 has increased from 15 to 22 Å in pH 4.0 by increasing the number of amino acids contributing to the α6-helix from 11 to 15, achieving a higher structural stability at pH 4.0. Taken together our experiments strongly suggest that the Mtb-DHBPS domain can transit between inactive monomer to active dimer depending upon its pH values, both in solution as well in crystal structure.
Dynamic properties of extremophilic subtilisin-like serine-proteases.
Venue: Journal of structural biology

The investigation of the structural determinants of enzymatic temperature adaptation is a crucial pre-requisite both in terms of fundamental research and industrial applications to develop new biocatalysts active at different temperature ranges. In several cases, the differences related to cold- or warm-adaptation are related to subtle structural and aminoacidic differences at the molecular level, often hard to detect. In this context, we present a comparative study of psychrophilic, mesophilic and thermophilic subtilisin-like serine proteases by all-atom molecular dynamics (MD) simulations in explicit solvent using a multiple-replica approach. Our results strongly enforce the current view on localized flexibility in crucial functional regions for cold-adapted serine proteases and point out a different optimization and usage of salt-bridge interactions and networks in cold- and warm-adapted enzymes. The analyses allow to identify a subset of structural and dynamic features strictly associated to cold adaptation and which change from cold- to heat-active subtilisins. In particular, the thermophilic subtilisin presents a high affinity calcium binding site which is not structurally conserved in the mesophilic and psychrophilic counterparts, which, as it turns out from the MD analyses, at the same position show a stable salt bridge network and no stabilizing intra-molecular interactions, respectively. These aspects, along with differential flexibility in regions close to the active site or substrate binding pocket, can be an indication of evolution at this protein site toward a lower stability moving from high to low temperature conditions.
Crystal structure of Sa240: a ribose pyranase homolog with partial active site from Staphylococcus aureus.
Venue: Journal of structural biology

Ribose is transported into cells in its pyranose form and must be rearranged to its furanose form for further utilization. Ribose pyranase RbsD catalyzes the conversion of ribose from the pyranose to furanose form. This is the key step for substrate supply to ribokinase RbsK, which converts ribose to ribose-5-phosphate for further metabolism. Sequence analysis indicated Sa240 from Staphylococcus aureus was a ribose pyranase homolog. Here we showed that Sa240 formed dimeric structure both in solution and in crystal. S240-ribose complex structure showed a ribose binding site formed by an incomplete active site compared with RbsD. Because the catalytic activity of ribose pyranase depends on its oligomeric state, we propose Sa240 is catalytically inactive in its dimeric structure.
Crystal structure of YdaL, a stand-alone small MutS-related protein from Escherichia coli.
Venue: Journal of structural biology

Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL₃₉-₁₇₅), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/β/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL₃₉-₁₇₅ protein were slightly lower than those of full-length YdaL₁-₁₈₇, suggesting that residues 1-38 may be involved in DNA binding.
Retrofit implementation of Zernike phase plate imaging for cryo-TEM.
Venue: Journal of structural biology

In-focus phase-plate imaging is particularly beneficial for cryo-TEM because it offers a substantial overall increase in image contrast, without an electron dose penalty, and it simplifies image interpretation. We show how phase-plate cryo-TEM can be implemented with an appropriate existing TEM, and provide a basic practical introduction to use of thin-film (carbon) phase plates. We point out potential pitfalls of phase-plate operation, and discuss solutions. We provide information on evaluating a particular TEM for its suitability.
The chiral structure of porous chitin within the wing-scales of Callophrys rubi.
Venue: Journal of structural biology

The structure of the porous three-dimensional reticulated pattern in the wing scales of the butterfly Callophrys rubi (the Green Hairstreak) is explored in detail, via scanning and transmission electron microscopy. A full 3D tomographic reconstruction of a section of this material reveals that the predominantly chitin material is assembled in the wing scale to form a structure whose geometry bears a remarkable correspondence to the srs net, well-known in solid state chemistry and soft materials science. The porous solid is bounded to an excellent approximation by a parallel surface to the Gyroid, a three-periodic minimal surface with cubic crystallographic symmetry I4₁32, as foreshadowed by Stavenga and Michielson. The scale of the structure is commensurate with the wavelength of visible light, with an edge of the conventional cubic unit cell of the parallel-Gyroid of approximately 310 nm. The genesis of this structure is discussed, and we suggest it affords a remarkable example of templating of a chiral material via soft matter, analogous to the formation of mesoporous silica via surfactant assemblies in solution. In the butterfly, the templating is achieved by the lipid-protein membranes within the smooth endoplasmic reticulum (while it remains in the chrysalis), that likely form cubic membranes, folded according to the form of the Gyroid. The subsequent formation of the chiral hard chitin framework is suggested to be driven by the gradual polymerisation of the chitin precursors, whose inherent chiral assembly in solution (during growth) promotes the formation of a single enantiomer.
Water permeability and characterization of aquaporin-11.
Venue: Journal of structural biology

The water permeability of aquaporin-11 (AQP11), which has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif in the water channel family, is controversial. Our previous study, however, showed that AQP11 is water permeable in proteoliposomes in which AQP11 molecules were reconstituted after purification with Fos-choline 10, which is the most suitable detergent available for stable solubilization of AQP11. In our previous study, we were unable to exclude the effect of the detergent on the water conductance. Therefore, in the present study, we measured the water permeability of AQP11 without detergent using vesicles that directly formed from Sf9 cell membranes expressing AQP11 molecules. The water permeability of AQP11 was 8-fold lower than that of AQP1 and 3-fold higher than that of mock-infected cell membrane, and was reversibly inhibited by mercury ions. Considering the slow but constant water permeable functions of AQP11, we performed homology modeling to search for a common structural feature. When comparing our model with those of other AQP structures, we found that Tyr83 facing the channel pore might be a key amino acid residue that decreases the water permeation of AQP11. Our findings indicate that AQP11 could be involved in slow but constant water movement across the membrane.
Nanoscale structure and mechanical behavior of growth lines in shell of abalone Haliotis gigantea.
Venue: Journal of structural biology

In the natural world, bottom-up hierarchical construction of complex structures results in materials with remarkable properties. A well known example is the nacre of mollusk shells, commonly called "mother of pearl", whose excellent strength and toughness has been the subject of research for many decades. A significant discovery has been the presence of periodic layers called "growth lines". These are thin distinct layers within the bulk of the shell which form periodically, with their structure affected by environmental changes. Studies of their formation and behavior offer valuable insight into the architecture of seashells. In this work, the structure and mechanical behavior of growth lines in shells of abalone Haliotis gigantea were investigated using electron microscopy and nanoindentation. Growth lines form directly out of nacre into layers of blocks and irregular particles. In comparison to nacre, they have basic structures, form rapidly, and are harder, which suggest that they serve a protective role during lifecycle transitions. This exemplifies how natural structures are able to closely control growth architecture in order to form different structures for different functions, all from the same base materials.
Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica).
Venue: Journal of structural biology

Luffin P1, the smallest ribosome-inactivating peptide from the seeds of Luffa cylindrica was found to have anti-HIV-1 activity in HIV-1 infected C8166 T-cell lines and be able to bind with HIV Rev Response Element. Nuclear magnetic resonance spectroscopy revealed that the Luffin P1 comprises a helix-loop-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Based on our findings, we conclude that unlike the well-studied ribosome-inactivating proteins, which exert their action through N-glycosidase activities, Luffin P1 demonstrates a novel inactivation mechanism probably through the charge complementation with viral or cellular proteins. Our work also provides a new scaffold for the design of novel inhibitors from a simple helical motif.
Dimer structure and conformational variability in the N-terminal region of an archaeal small heat shock protein, StHsp14.0.
Venue: Journal of structural biology

Small heat shock proteins (sHsps), which are categorized into a class of molecular chaperones, bind and stabilize denatured proteins to prevent aggregation. The sHsps undergo transition between different oligomeric states to control their hydrophobicity. So far, only the structures of sHsps in large oligomeric states have been reported. Here we report the structure of StHsp14.0 from Sulfolobus tokodaii in the dimeric state, which is formed by means of a mutation at the C-terminal IXI/V motif. The dimer is the sole building block in two crystal forms, and the dimeric mode is the same as that in the large oligomers. The N-terminal helix has variety in its conformation. Furthermore, spectroscopic and biochemical experiments were performed to investigate the conformational variability at the N-terminus. The structural, dynamical and oligomeric properties suggest that chaperone activity of StHsp14.0 is mediated by partially dissolved oligomers.
Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM.
Venue: Journal of structural biology

Electron microscopy at a resolution of 0.4nm or better requires more careful adjustment of the illumination than is the case at a resolution of 0.8nm. The use of current-axis alignment is not always sufficient, for example, to avoid the introduction of large phase errors, at higher resolution, due to axial coma. In addition, one must also ensure that off-axis coma does not corrupt the data quality at the higher resolution. We particularly emphasize that the standard CTF correction does not account for the phase error associated with coma. We explain the cause of both axial coma and the typically most troublesome component of off-axis coma in terms of the well-known shift of the electron diffraction pattern relative to the optical axis that occurs when the illumination is not parallel to the axis. We review the experimental conditions under which coma causes unacceptably large phase errors, and we discuss steps that can be taken when setting up the conditions of illumination, so as to ensure that neither axial nor off-axis coma is a problem.
Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase.
Venue: Journal of structural biology

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.
Binding, reconstitution and 2D crystallization of membrane or soluble proteins onto functionalized lipid layer observed in situ by reflected light microscopy.
Venue: Journal of structural biology

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface.
Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin.
Venue: Journal of structural biology

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.
Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II.
Venue: Journal of structural biology

Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.
Structural insight into the glycosylphosphatidylinositol transamidase subunits PIG-K and PIG-S from yeast.
Venue: Journal of structural biology

The addition of glycosylphosphatidylinositol (GPI) anchors to eukaryotic proteins in the lumen of the endoplasmic reticulum is catalyzed by the transamidase complex, composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here PIG-K(24-337) and PIG-S(38-467) from yeast, including the residues 24-337 and 38-467 of the entire 411 and 534 residue protein, respectively, was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that yPIG-K(24-377) comprises 52% α-helix and 12% β-sheet, whereas yPIG-S(38-467) involves 58% α-helix and 18% β-sheet. The radius of gyration (R(g)) and the maximum size (D(max)) of both proteins have been analyzed by small angle X-ray scattering (SAXS) and determined to be 2.64±0.3 and 10.3±0.1 nm (yPIG-K(24-377)) as well as 3.06±0.02 nm (R(g)) and 16.9±0.4 nm (D(max)) in the case of yPIG-S(38-467), respectively. Using an ab initio approach, the first low-resolution solution structures of both proteins were restored. yPIG-K(24-377) is an elongated particle consisting of an egg-like portion and a small globular segment linked together by an 1.9 nm long stalk. yPIG-S(38-467) forms an elongated molecule in solution with a larger domain of 10.1 nm in length, a diameter of 9.1 nm and a smaller domain of 6.7 nm in length and 3.4 nm in width. The two domains of yPIG-S(38-467) are tilted relative to each other. Finally, the arrangements of PIG-K and PIG-S inside the ensemble of the transamidase complex are discussed.
Self-aligning amelogenin nanoribbons in oil-water system.
Venue: Journal of structural biology

The highly organized microstructure of dental enamel is a result of protein-guided anisotropic growth of apatite nanofibers. It is established that amelogenin proteins, the main constituent of the developing enamel matrix, form nanospheres in vitro, but the amphiphilic nature of the full-length protein conveys the possibility of generating more complex structures as observed with other surfactant-like molecules. This study tested if the use of metastable oil-water emulsions can induce supramolecular assemblies of amelogenin. Recombinant full-length amelogenin, rH174, was mixed into octanol/ethyl acetate preparations of different ratios to form emulsions at pH 4.5 and 7.4. Atomic force and electron microscopy showed the formation of 16.7±1.0nm wide nanoribbons which grew to several micrometer length over a period of days. Nanoribbons formed from reverse micelles by enabling hydrophobic tails of the molecules to interact while preventing the formation of amelogenin nanospheres. Ribbon formation required the presence of calcium and phosphate ions and may be localized at a dark central line along the amelogenin ribbons. The ribbons have a strong tendency to align in parallel maintaining 5-20nm space between each other. The growth rates and number of ribbons were significantly higher at pH 4.5 and related to the metastability of the emulsion. A model for ribbon extension proposes the addition of short segments or amelogenin dimers to the ends of the ribbon. The formation of self-aligning and uniaxially elongating amelogenin structures triggered by the presence of calcium and phosphate may represent a suitable new model for protein controlled mineralization in enamel.