nils-herrmann/clean_scirepeval huggingface dataset:
Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction.
Venue: Journal of structural biology

The autophagy-related proteins are thought to serve multiple functions in Plasmodium and are considered essential to parasite survival and development. We have studied two key interacting proteins, Atg8 and Atg3, of the autophagy pathway in Plasmodium falciparum. These proteins are vital for the formation and elongation of the autophagosome and essential to the process of macroautophagy. Autophagy may be required for conversion of the sporozoite into erythrocytic-infective merozoites and may be crucial for other functions during asexual blood stages. Here we describe the identification of an Atg8 family interacting motif (AIM) in Plasmodium Atg3, which binds Plasmodium Atg8. We determined the co-crystal structure of PfAtg8 with a short Atg3¹⁰³⁻¹¹⁰ peptide, corresponding to this motif, to 2.2 Å resolution. Our in vitro interaction studies are in agreement with our X-ray crystal structure. Furthermore they suggest an important role for a unique Apicomplexan loop absent from human Atg8 homologues. Prevention of the protein-protein interaction of full length PfAtg8 with PfAtg3 was achieved at low micromolar concentrations with a small molecule, 1,2,3-trihydroxybenzene. Together our structural and interaction studies represent a starting point for future antimalarial drug discovery and design for this novel protein-protein interaction.
The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion.
Venue: Journal of structural biology

Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.
Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor.
Venue: Journal of structural biology

Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases. As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7Å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg(11) at P3 position, slightly reduced interactions at the prime side (Pn') of the primary binding loop and at the secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological applications.
Quantitative analysis of spin exchange interactions to identify β strand and turn regions in Ure2 prion domain fibrils with site-directed spin labeling.
Venue: Journal of structural biology

Amyloid formation is associated with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register β structure. With quantitative analysis of spin exchange interactions, we show that residues 8-12 form the first β strand, followed by a turn at residues 13-14, and then the second β strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of β-strand and turn regions in this work suggests that quantitative analysis of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the β-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.
The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase--rational design of mutants and the crystal structures of the A85H and W104Y variants.
Venue: Journal of structural biology

Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected.
Crystal structure of 1,3Gal43A, an exo-β-1,3-galactanase from Clostridium thermocellum.
Venue: Journal of structural biology

Glycoside hydrolase family 43 (GH43) consists of a variety of enzymes distributed widely in prokaryotes and eukaryotes. The mechanism by which GH43 enzymes hydrolyze oligosaccharides requires three essential acidic amino acid residues. However, one of them is thought to be missing in galactan β-1,3-galactosidases from the GH43 family. Ct1,3Gal43A, from Clostridium thermocellum, is comprised of a GH43 domain, a CBM13 domain, and a dockerin domain and exhibits an unusual ability to hydrolyze β-1,3-galactan in the presence of a β-1,6 linked branch. Here, we present its crystal structure at 2.7 Å resolution and complex structures of the enzyme with several substrates and analogs. Two modes of substrate binding were observed at the β site of the CtCBM13 domain, and one galactobiose molecule was found in an "L" shaped pocket of the CtGH43 domain, which appears large enough to accommodate two more galactose units. In addition, we found that mutating Glu112 to Gln or Ala eliminated the galactan hydrolysis activity of Ct1,3Gal43A while did not disrupt its ligand binding ability. Combining this results and the crystal structure we identified Glu112 in Ct1,3Gal43A as the 'missing' essential acidic residue in galactan β-1,3-galactosidases. Structural information presented here also suggests a mechanism by which Ct1,3Gal43A bypasses β-1,6 linked branches in the substrate and another mechanism by which the substrate is delivered 'in trans' from the CBM13 domain to the catalytic GH43 domain.
Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy.
Venue: Journal of structural biology

Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.
Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM).
Venue: Journal of structural biology

In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future.
Sea urchin tooth mineralization: calcite present early in the aboral plumula.
Venue: Journal of structural biology

In both vertebrate bone, containing carbonated hydroxyapatite as the mineral phase, and in invertebrate hard tissue comprised of calcium carbonate, a popular view is that the mineral phase develops from a long-lived amorphous precursor which later transforms into crystal form. Important questions linked to this popular view are: when and where is the crystallized material formed, and is amorphous solid added subsequently to the crystalline substrate? Sea urchin teeth, in which the earliest mineral forms within isolated compartments, in a time and position dependent manner, allow direct investigation of the timing of crystallization of the calcite primary plates. Living teeth of the sea urchin Lytechinus variegatus, in their native coelomic fluid, were examined by high-energy synchrotron X-ray diffraction. The diffraction data show that calcite is present in the most aboral portions of the plumula, representing the very earliest stages of mineralization, and that this calcite has the same crystal orientation as in the more mature adoral portions of the same tooth. Raman spectroscopy of the aboral plumula confirms the initial primary plate mineral material is calcite and does not detect amorphous calcium carbonate; in the more mature adoral incisal flange, it does detect a broader calcite peak, consistent with two or more magnesium compositions. We hypothesize that some portion of each syncytial membrane in the plumula provides the information for nucleation of identically oriented calcite crystals that subsequently develop to form the complex geometry of the single crystal sea urchin tooth.
Structures of amyloid fibrils formed by the prion protein derived peptides PrP(244-249) and PrP(245-250).
Venue: Journal of structural biology

While the formation of amyloid fibrils from diverse peptide and protein sequences is well established, the molecular determinants of structure and assembly are not well understood. In particular, the relationship between amino acid sequence and the type of internal steric zipper packing adopted in amyloid fibrils has not been established. Here we report the structures of two cytotoxic amyloid peptides derived from the mammalian prion protein, PrP(244-249) and PrP(245-250), determined using solid state NMR. While the amino acid composition of these two hexapeptides is very similar (ISFLIF and SFLIFL), the intermolecular interactions that give rise to the intersheet packing within the fibrils differ significantly. PrP(245-250) adopts a class 1 steric zipper, with parallel sheets stacked in an antiparallel face to face arrangement, stabilized by N- to C-terminal salt bridges. PrP(244-249), by contrast, forms two different intersheet interfaces within amyloid fibrils, with parallel opposing sheets in either a face to face (class 3) or face to back (class 2) arrangement. The fibrils formed by this peptide are primarily stabilized by close packing of the hydrophobic side chains, with contributions from side-chain to backbone hydrogen bonding (class 2 only). Thus, the structures presented here provide new insight into the relationship between amino acid sequence and the types of interactions stabilizing amyloid fibrils.
Molecular Modelling and Molecular Dynamics studies of GD1A, GD1B and their complexes with BoNT/B--perspectives in interaction and specificity.
Venue: Journal of structural biology

The conformational property for the oligosaccharide structure of GD1A and GD1B in aqueous environment is studied by 10 ns Molecular Dynamics simulation using all atom model. Based on the trajectory analysis four conformational models are proposed for GD1A and one for GD1B. Direct and water mediated hydrogen bonding interactions plays a prominent role in stabilizing these conformational structures. The Molecular Modelling and 10 ns MD simulation of Botulinum Neuro Toxin/B-GD1A and BoNT/B-GD1B complex revealed that this toxin can interact with GD1A in the single binding mode and with GD1B in two binding modes. Least mobility is seen for GD1A in the binding pocket of BoNT/B. The GTSM comparison, pair interaction energy calculation, total energy calculation, MM/PBSA binding free energy calculation and RMSD predicts that GD1A is a better receptor for BoNT/B compared to GD1B. The internal NeuNAc1 tends to form more than 70% of hydrogen bonds with BoNT/B both in GD1A and GD1B, hence specifying this particular site as a crucial space for the therapeutic design that can restrict the pathogenic activity of BoNT/B.
Inter-phylum structural conservation of the magnetosome-associated TPR-containing protein, MamA.
Venue: Journal of structural biology

Magnetotactic bacteria enclose the magnetosome, a unique prokaryotic sub-cellular organelle that allows the biomineralization of magnetic nano-crystals. Membrane-coated magnetosomes are arranged into a linear chain that permits magnetotactic bacteria to navigate geomagnetic fields. Magnetosome assembly and biomineralization are controlled by conserved magnetosome-associated proteins, including MamA, a tetra-trico-peptide repeat (TPR)-containing protein that was shown to coat the magnetosome membrane. In this study, two MamA structures from Candidatus Magnetobacterium bavaricum (Mbav) were determined via X-ray crystallography. These structures confirm that Mbav MamA folds as a sequential TPR protein and shares a high degree of structural similarity with homologous MamA proteins from Magnetospirillum species. Furthermore, the two TPR-containing domains of MamA are separated by an interphylum-conserved region containing a flexible hinge that is involved in ligand binding and recognition. Finally, substantial differences were found in the local stabilization of the MamA N-terminal domain as a result of the loss of an evolutionary conserved salt bridge.
SIMPLE: Software for ab initio reconstruction of heterogeneous single-particles.
Venue: Journal of structural biology

The open source software suite SIMPLE: Single-particle IMage Processing Linux Engine provides data analysis methods for single-particle cryo-electron microscopy (cryo-EM). SIMPLE addresses the problem of obtaining 3D reconstructions from 2D projections only, without using an input reference volume for approximating orientations. The SIMPLE reconstruction algorithm is tailored to asymmetrical and structurally heterogeneous single-particles. Its basis is global optimization with the use of Fourier common lines. The advance that enables ab initio reconstruction and heterogeneity analysis is the separation of the tasks of in-plane alignment and projection direction determination via bijective orientation search - a new concept in common lines-based strategies. Bijective orientation search divides the configuration space into two groups of paired parameters that are optimized separately. The first group consists of the rotations and shifts in the plane of the projection; the second group consists of the projection directions and state assignments. In SIMPLE, ab initio reconstruction is feasible because the 3D in-plane alignment is approximated using reference-free 2D rotational alignment. The subsequent common lines-based search hence searches projection directions and states only. Thousands of class averages are analyzed simultaneously in a matter of hours. Novice SIMPLE users get a head start via the well documented front-end. The structured, object-oriented back-end invites advanced users to develop new alignment and reconstruction algorithms. An overview of the package is presented together with benchmarks on simulated data. Executable binaries, source code, and documentation are available at http://simple.stanford.edu.
Explorations of linked editosome domains leading to the discovery of motifs defining conserved pockets in editosome OB-folds.
Venue: Journal of structural biology

Trypanosomatids form a group of protozoa which contain parasites of human, animals and plants. Several of these species cause major human diseases, including Trypanosoma brucei which is the causative agent of human African trypanosomiasis, also called sleeping sickness. These organisms have many highly unusual features including a unique U-insertion/deletion RNA editing process in the single mitochondrion. A key multi-protein complex, called the ∼20S editosome, or editosome, carries out a cascade of essential RNA-modifying reactions and contains a core of 12 different proteins of which six are the interaction proteins A1 to A6. Each of these interaction proteins comprises a C-terminal OB-fold and the smallest interaction protein A6 has been shown to interact with four other editosome OB-folds. Here we report the results of a "linked OB-fold" approach to obtain a view of how multiple OB-folds might interact in the core of the editosome. Constructs with variants of linked domains in 25 expression and co-expression experiments resulted in 13 soluble multi-OB-fold complexes. In several instances, these complexes were more homogeneous in size than those obtained from corresponding unlinked OB-folds. The crystal structure of A3(OB) linked to A6 could be elucidated and confirmed the tight interaction between these two OB domains as seen also in our recent complex of A3(OB) and A6 with nanobodies. In the current crystal structure of A3(OB) linked to A6, hydrophobic side chains reside in well-defined pockets of neighboring OB-fold domains. When analyzing the available crystal structures of editosome OB-folds, it appears that in five instances "Pocket 1" of A1(OB), A3(OB) and A6 is occupied by a hydrophobic side chain from a neighboring protein. In these three different OB-folds, Pocket 1 is formed by two conserved sequence motifs and an invariant arginine. These pockets might play a key role in the assembly or mechanism of the editosome by interacting with hydrophobic side chains from other proteins.