Optimizing protein crosslinking control: Synergistic quenching effects of glycine, histidine, and lysine on glutaraldehyde reactions.

Glutaraldehyde (GA) is a protein crosslinker widely used in biochemical and pharmaceutical research because it can rapidly stabilize and immobilize substrates via amine group interactions. However, controlling GA crosslinking is challenging owing to its swift reactivity and the influence of various solution conditions, such as pH and concentrations of the substrate and crosslinker. Although extensive research has focused on GA cross-linking mechanisms, studies on quenching, which is critical for preventing non-specific aggregation during prolonged storage, remain sparse. This study examines the quenching efficiency of a combined amino acid mixture of glycine, histidine, and lysine, which are commonly used as individual quenchers. Our findings, confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, demonstrate that this amino acid blend offers superior quenching compared to single amino acids, enhancing quenching activity across a wide pH spectrum. These results provide a novel approach for mitigating the high reactivity of GA with implications for improving sample preservation and stabilization in a range of biochemical applications, including microscopy and cell fixation.

Comparative Analysis of T4SS Molecular Architectures.

The recently published high-resolution R388 T4SS structure provides exciting new details about the complete complex of T4SS, including the components making up the stalk and arches, numerous symmetry mismatches between regions of the complex, and an intriguing interpretation of the closed stalk and radial symmetry of the inner membrane complex, which is related to pilus biogenesis assembly. However, there are a few unidentified densities in the electron microscopy map and portions of the identified component sequences for which the structure is not yet known. It is also unclear how well this minimized DNA-transporting T4SS predicts the structure of other T4SSs, such as expanded systems and those that transport proteins rather than DNA. In this review, we evaluate what can be inferred from the recent high-resolution structure of the R388 T4SS with respect to the Cag and Dot/Icm systems. These systems were selected because, given what is currently known about these systems, we expect them to present most structural differences compared to the R388 T4SS structure. Furthermore, we discuss bacterial physiology and diversity, the T4SS structures and their variations between different bacterial species. These insights may prove beneficial for researchers who elucidate the structure and functions of T4SS in different bacterial species.

Studies of functional properties of espin 1: Its interaction to actin filaments.

Actin is a multifunctional biomolecule that forms not only basic structural bodies such as filopodia and lamellipodia, but also large microvilli-like organelles like stereocilia. Actin consists of four sub-domains (S1, S2, S3, and S4), and the "target-binding groove" formed between S1 and S3 is the major binding site for various actin binding proteins. Actin filament dynamics are regulated by numerous actin binding proteins with different mechanisms of actin binding, assembly, and disassembly such as actin severing, branching, and bundling. Ectoplasmic specialization protein 1 (espin 1) is an actin binding and bundling protein that is specifically implicated in the elongation and stabilization of stereocilia as a binding partner with myosin III. However, little is known about the molecular structure, actin bundling, and stabilizing mechanism of espin 1; hence, we investigated the interaction between actin and espin 1 through structural data. In this study, we first purified human espin 1 in an E. coli system following a new detergent-free approach and then demonstrated the 2D structure of full-length espin 1 using transmission electron microscopy along with Nickel nitrilotriacetic acid nanogold labeling and 2D averaging using SPIDER. Furthermore, we also determined the espin 1 binding domain of actin using a co-sedimentation assay along with gelsolin and myosin S1. These findings are not only beneficial for understanding the actin binding and bundling mechanism of espin 1, but also shed light on its elongation, stabilization, and tip-localization mechanisms with myosin III. This study thus provides a basis for understanding the molecular structure of espin 1 and can contribute to various hearing-related diseases, such as hearing loss and vestibular dysfunction.

Artificial Intelligence in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) has become an unrivaled tool for determining the structure of macromolecular complexes. The biological function of macromolecular complexes is inextricably tied to the flexibility of these complexes. Single particle cryo-EM can reveal the conformational heterogeneity of a biochemically pure sample, leading to well-founded mechanistic hypotheses about the roles these complexes play in biology. However, the processing of increasingly large, complex datasets using traditional data processing strategies is exceedingly expensive in both user time and computational resources. Current innovations in data processing capitalize on artificial intelligence (AI) to improve the efficiency of data analysis and validation. Here, we review new tools that use AI to automate the data analysis steps of particle picking, 3D map reconstruction, and local resolution determination. We discuss how the application of AI moves the field forward, and what obstacles remain. We also introduce potential future applications of AI to use cryo-EM in understanding protein communities in cells.

Structural Analysis of Human Fascin-1: Essential Protein for Actin Filaments Bundling.

Fascin, a major actin cross-linking protein, is expressed in most vertebrate epithelial tissues. It organizes actin filaments into well-ordered bundles that are responsible for the extension of dynamic membrane protrusions, including microspikes, filopodia, and invadopodia from cell surfaces, which are involved in cell migration and invasion as critical components of cancer metastasis. However, it is not well-understood how fascin-1 induces actin binding/bundling and where fascin-1 localizes along the actin filaments, thus facilitating actin bundle formation. In the present study, we attempted to clarify these problems by using biochemical and electron microscopic analyses using various fascin-1 constructs. Three dimensional structures of actin/fascin-1 complex were obtained by electron microscopy (EM) with iterative helical real-space reconstruction (IHRSR) and tomography. We revealed that the N-terminal region containing the Actin-Binding Site 2 (ABS2) of fascin-1 is responsible for actin bundling and the C-terminal region is important for the dimerization of fascin-1. We also found that the dimerization of fascin-1 through intermolecular interactions of the C-terminal region is essential for actin bundling. Since fascin is an important factor in cancer development, it is expected that the findings of present study will provide useful information for development of therapeutic strategies for cancer.

Molecular architecture of the human caveolin-1 complex.

Membrane-sculpting proteins shape the morphology of cell membranes and facilitate remodeling in response to physiological and environmental cues. Complexes of the monotopic membrane protein caveolin function as essential curvature-generating components of caveolae, flask-shaped invaginations that sense and respond to plasma membrane tension. However, the structural basis for caveolin's membrane remodeling activity is currently unknown. Here, we show that, using cryo-electron microscopy, the human caveolin-1 complex is composed of 11 protomers organized into a tightly packed disc with a flat membrane-embedded surface. The structural insights suggest a previously unrecognized mechanism for how membrane-sculpting proteins interact with membranes and reveal how key regions of caveolin-1, including its scaffolding, oligomerization, and intramembrane domains, contribute to its function.

Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS.

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

Structural analysis of the Legionella pneumophila Dot/Icm type IV secretion system core complex.

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires' Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.

Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex.

The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing five proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.

Dual Binding to Orthosteric and Allosteric Sites Enhances the Anticancer Activity of a TRAP1-Targeting Drug.

The molecular chaperone TRAP1 is the mitochondrial paralog of Hsp90 and is overexpressed in many cancer cells. The orthosteric ATP-binding site of TRAP1 has been considered the primary inhibitor binding location, but TRAP1 allosteric modulators have not yet been investigated. Here, we generated and characterized the Hsp90 inhibitor PU-H71, conjugated to the mitochondrial delivery vehicle triphenylphosphonium (TPP) with a C10 carbon spacer, named SMTIN-C10, to enable dual binding to orthosteric and allosteric sites. In addition to tight binding with the ATP-binding site through the PU-H71 moiety, SMTIN-C10 interacts with the E115 residue in the N-terminal domain through the TPP moiety and subsequently induces structural transition of TRAP1 to a tightly packed closed form. The data indicate the existence of a druggable allosteric site neighboring the orthosteric ATP pocket that can be exploited to develop potent TRAP1 modulators.

Structure of the Helicobacter pylori Cag type IV secretion system.

Bacterial type IV secretion systems (T4SSs) are molecular machines that can mediate interbacterial DNA transfer through conjugation and delivery of effector molecules into host cells. The Helicobacter pylori Cag T4SS translocates CagA, a bacterial oncoprotein, into gastric cells, contributing to gastric cancer pathogenesis. We report the structure of a membrane-spanning Cag T4SS assembly, which we describe as three sub-assemblies: a 14-fold symmetric outer membrane core complex (OMCC), 17-fold symmetric periplasmic ring complex (PRC), and central stalk. Features that differ markedly from those of prototypical T4SSs include an expanded OMCC and unexpected symmetry mismatch between the OMCC and PRC. This structure is one of the largest bacterial secretion system assemblies ever reported and illustrates the remarkable structural diversity that exists among bacterial T4SSs.

The actin bundling activity of actin bundling protein 34 is inhibited by calcium binding to the EF2.

Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.

Analysis of nano-crystals: Evaluation of heavy metal-embedded biological specimen by high voltage electron microscopy.

Heavy metal compounds are adsorbed onto biological specimen in order to enhance the contrast as well as to preserve the structural features of the specimen against electron beam-induced radiation damage. In particular, in combination with computational image processing, negative staining is widely used for structural analysis of protein complexes to moderate resolutions. Image analysis of negatively stained biological specimen is known to suffer from limited achievable resolution due to dehydration and large grain size of staining molecules although the extent of such effect remains somewhat dubious. Stain molecules exist as grains under electron beam. However, clear observation of the crystalline nature of the grains and their association with biological specimen has not been thoroughly demonstrated. In this study, we attempted high-resolution TEM (HRTEM) using high voltage electron microscopy and electron crystallography analysis for the detailed characterization of negatively stained biological specimen, focusing on physical state and chemical composition of the stain molecules. The electron crystallography analysis allowed for the identification of the crystal constituents of widely used stains, hence revealing the chemical nature and the morphology of the stain molecules at specimen level. This study re-evaluated generally accepted notions on negative staining, and may help correctly interpreting the structural analysis of stained biological specimen.

Characterisation of conformational and functional features of alkyl hydroperoxide reductase E-like protein.

Alkyl hydroperoxide reductase E (AhpE) is a member of the peroxidase family of enzymes that catalyse the reduction of peroxides, however its structural and functional roles are still unclear in details. In this study, we used the Thermococcus kodakarensis AhpE-like protein as a model to investigate structure-function relationships including the molecular properties of DNA binding activity. Multiple sequence alignment, structural comparison and biochemical analyses revealed that TkAhpE includes conserved peroxidase residues in the active site, and exhibits peroxidase activity with structure-dependent holdase chaperone function. Following electrophoretic mobility shift assays and electron microscopy analysis demonstrated distinctive binding features of TkAhpE to the DNA showing that their dimeric conformer can bind to the double-stranded DNA, but not to the single-stranded DNA, indicating its striking molecular features to double-stranded DNA-specific interactions. Based on our results, we provided that TkAhpE is a multifunctional peroxidase displaying structure-dependent molecular chaperone and DNA binding activities.

Site-specific mutagenesis of yeast 2-Cys peroxiredoxin improves heat or oxidative stress tolerance by enhancing its chaperone or peroxidase function.

Yeast peroxiredoxin II (yPrxII) is an antioxidant enzyme that plays a protective role against the damage caused by reactive oxygen species (ROS) in Saccharomyces cerevisiae. This enzyme consists of 196 amino acids containing 2-Cys Prx with highly conserved two active cysteine residues at positions 48 and 171. The yPrxII has dual enzymatic functions as a peroxidase and molecular chaperone. To understand the effect of additional cysteine residues on dual functions of yPrxII, S79C-yPrxII and S109C-yPrxII, the substitution of Ser with Cys residue at 79 and 109 positions, respectively, was generated. S109C-yPrxII and S79C-yPrxII showed 3.7- and 2.7-fold higher chaperone and peroxidase activity, respectively, than the wild type (WT). The improvement in enzyme activity was found to be closely associated with structural changes in proteins. S109C-yPrxII had increased ß-sheet in its secondary structure and formed high-molecular-weight (HMW) as well as low-molecular-weight (LMW) complexes, but S79C-yPrxII formed only LMW complexes. HMW complexes predominantly exhibited a chaperone function, and LMW complexes showed a peroxidase function. In addition, transgenic yeast cells over-expressing Cys-substituted yPrxII showed greater tolerance against heat and oxidative stress compared to WT-yPrxII.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.

Technical Advances in Intracellular Detection Using Immuno-Gold Particles: Simple Cryofixation with Metal Contact Quick Freezing.

The preparation of biological specimens using cryofixation techniques ensures excellent visibility of intracellular structures and preserves the antigenic sites of subcellular molecules. Hence, cryofixation is an effective method of preparing samples for analyses using antibodies conjugated to gold nanoparticles that are designed to detect the localization of specific target molecules within cells. However, cryofixation cannot be utilized easily because it requires expensive equipment and skilled technologists, resulting in a high level of expense for researchers. Here, we describe a simple technical approach to cryofixation that uses metal contact quick freezing followed by a modified freeze substitution technique and immuno-gold labeling electron microscopy. Micrograph images of cells prepared using this modified cryofixation method demonstrated its superiority over chemical fixation for high contrast visualization of the morphologies of cellular components and preservation of antigenicity for immuno-gold labeling. This report provides valuable technical information related to the advancement of metal contact quick freezing techniques, which can be used to visualize biomedical events of interest in an easy, simple, and rapid manner.

Molecular Insights into Toluene Sensing in the TodS/TodT Signal Transduction System.

TodS is a sensor kinase that responds to various monoaromatic compounds, which either cause an agonistic or antagonistic effect on phosphorylation of its cognate response regulator TodT, and controls tod operon expression in Pseudomonas putida strains. We describe a molecular sensing mechanism of TodS that is activated in response to toluene. The crystal structures of the TodS Per-Arnt-Sim (PAS) 1 sensor domain (residues 43-164) and its complex with toluene (agonist) or 1,2,4-trimethylbenzene (antagonist) show a typical ß2α3ß3 PAS fold structure (residues 45-149), forming a hydrophobic ligand-binding site. A signal transfer region (residues 150-163) located immediately after the canonical PAS fold may be intrinsically flexible and disordered in both apo-PAS1 and antagonist-bound forms and dramatically adapt an α-helix upon toluene binding. This structural change in the signal transfer region is proposed to result in signal transmission to activate the TodS/TodT two-component signal transduction system. Site-directed mutagenesis and ß-galactosidase assays using a P. putida reporter strain system verified the essential residues involved in ligand sensing and signal transfer and suggest that the Phe(46) residue acts as a ligand-specific switch.

New insight into multifunctional role of peroxiredoxin family protein: Determination of DNA protection properties of bacterioferritin comigratory protein under hyperthermal and oxidative stresses.

Bacterioferritin comigratory protein (BCP) is a monomeric conformer acting as a putative thiol-dependent bacterial peroxidase, however molecular basis of DNA-protection via DNA-binding has not been clearly understood. In this study, we characterized the DNA binding properties of BCP using various lengths and differently shaped architectures of DNA. An electrophoretic mobility shift assay and electron microscopy analysis showed that recombinant TkBCP bound to DNA of a circular shape (double-stranded DNA and single-stranded DNA) and a linear shape (16-1000 bp) as well as various architectures of DNA. In addition, DNA protection experiments indicated that TkBCP can protect DNA against hyperthermal and oxidative stress by removing highly reactive oxygen species (ROS) or by protecting DNA from thermal degradation. Based on these results, we suggest that TkBCP is a multi-functional DNA-binding protein which has DNA chaperon and antioxidant functions.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.