Structural Insights Research Articles

Structural insights into the role of the prosegment binding loop in a papain-superfamily cysteine protease from Treponema denticola.

Periodontal diseases afflict 20-50% of the global population and carry serious health and economic burdens. Chronic periodontitis is characterized by inflammation of the periodontal pocket caused by dysbiosis. This dysbiosis is coupled with an increase in the population of Treponema denticola, a spirochete bacterium with high mobility and invasivity mediated by a number of virulence factors. One such virulence factor is TDE0362, a multidomain protein with a carboxy-terminal papain-superfamily cysteine protease (C0362). Most papain-superfamily cysteine proteases are produced as proenzymes with a prodomain that interacts with the prosegment binding loop (PBL), requiring proteolytic processing for full activation. Previous studies have indicated that C0362 is not produced as a proenzyme, suggesting an alternative regulatory mechanism. We previously determined the crystal structure of C0362 captured in an inactive conformation with an oxidized catalytic cysteine and a disordered PBL. In this follow-up study, we evaluated the active-site architecture and the PBL in two mutant (Y559A and C412S) structures and an inhibitor-bound (E64) structure to provide insight into the role that the PBL plays in the generation of active enzyme. Our results implicate Tyr559 as playing a critical role in the transition of the enzyme to an active state. We subsequently utilized the structural information to generate models of C0362 bound to human complement factors C3 and C4. Collectively, our results provide insight into the regulatory mechanism and putative substrate-binding interfaces of C0362, highlighting avenues of further research towards inhibition of this essential virulence factor.

Biochemical and structural insights into pinoresinol hydroxylase from Pseudomonas sp.

The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH’s conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members.

Biased signaling in GPCRs: Structural insights and implications for drug development.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans, playing a crucial role in regulating diverse cellular processes and serving as primary drug targets. Traditional drug design has primarily focused on ligands that uniformly activate or inhibit GPCRs. However, the concept of biased agonism-where ligands selectively stabilize distinct receptor conformations, leading to unique signaling outcomes-has introduced a paradigm shift in therapeutic development. Despite the promise of biased agonists to enhance drug efficacy and minimize side effects, a comprehensive understanding of the structural and biophysical mechanisms underlying biased signaling is essential. Recent advancements in GPCR structural biology have provided unprecedented insights into ligand binding, conformational dynamics, and the molecular basis of biased signaling. These insights, combined with improved techniques for characterizing ligand efficacy, have driven the development of biased ligands for several GPCRs, including opioid, angiotensin, and adrenergic receptors. This review synthesizes these developments, from mechanisms to drug discovery in biased signaling, emphasizing the role of structural insights in the rational design of next-generation biased agonists with superior therapeutic profiles. Ultimately, these advances hold the potential to revolutionize GPCR-targeted drug discovery, paving the way for more precise and effective treatments.

Advanced Study of Structural and Optical Characteristics of Nanocrystalline Coomassie Brilliant Blue G-250 for Optoelectronic Device Optimization

This study explores the structural and optical properties of Coomassie Brilliant Blue G-250 (CBBG) using X-ray diffraction (XRD) and spectroscopic techniques. XRD analysis of both powder and thin film samples reveals distinct structural features: the powder exhibits a polycrystalline structure with strong diffraction peaks corresponding to cubic lattice planes, while the thin film shows a mix of crystalline orientations within an amorphous matrix. The Williamson-Hall relation is applied to determine average crystallite sizes, yielding 85.5nm for the powder and 71.8nm for the thin film, indicating their nanocrystalline nature. Microstrain analysis also reveals compressive strain within the thin film. These structural insights are critical for understanding CBBG's mechanical and optical properties, vital for biochemical and optoelectronic applications. Density functional theory (DFT) calculations with the B3LYP/6-311++G(d,p) basis set were used to optimize the molecular geometry and analyze HOMO-LUMO energies and reactive sites via the MEP map. CBBG shows higher hyperpolarizability than urea, indicating its potential for nonlinear optical applications. Miller indices further support its optoelectronic optimization. The real (ε1) and imaginary (ε2) parts of the dielectric constant were analyzed, revealing CBBG thin films exhibit strong polarization and charge displacement, influenced by structural disorder. Additionally, the volume and surface energy loss functions (VELF and SELF) highlight significant internal energy dissipation, emphasizing CBBG's potential for optoelectronic devices. The complex optical conductivity analysis reveals key absorption and dispersion behaviors essential for advanced photonic and electronic device design.

Mutational study of a lytic polysaccharide monooxygenase from Myceliophthora thermophila (MtLPMO9F): Structural insights into substrate specificity and regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the biotechnological exploitation of lignocellulosic biomass, yet their efficient application depends on the in-depth understanding of their mechanism of action. Here, we describe the structural and mutational characterization of a C4-active LPMO from Myceliophthora thermophila, MtLPMO9F, that belongs to auxiliary activity family 9 (AA9). MtLPMO9F is active on cellulose, cello-oligosaccharides and xyloglucan. The crystal structure of MtLPMO9F catalytic domain, determined at 2.3Å resolution, revealed a double conformation for loop L3 with a potential implication in the formation of aglycon subsites. Product analysis of reactions with cello-oligosaccharides showed a prevalent -4 to +2 binding mode. Subsequent biochemical characterization of 4 MtLPMO9F point mutants further provided insights in LPMO structure-function relationships regarding both substrate binding and the role of second-coordination sphere residues.

Fully graphic degree sequences and P-stable degree sequences

The notion of P-stability of an infinite set of degree sequences plays influential role in approximating the permanents, rapidly sampling the realizations of graphic degree sequences, or even studying and improving network privacy. While there exist several known sufficient conditions for P-stability, we don't know any useful necessary condition for it. We also do not have good insight of possible structure of P-stable degree sequence families.At first we will show that every known infinite P-stable degree sequence set, described by inequalities of the parameters n,c1,c2,Σ (the sequence length, the maximum and minimum degrees and the sum of the degrees) is “fully graphic” meaning that every degree sequence from the region with an even degree sum, is graphic. Furthermore, if Σ does not occur in the determining inequality, then the notions of P-stability and full graphicality will be proved equivalent. In turn, this equality provides a strengthening of the well-known theorem of Jerrum, McKay and Sinclair about P-stability, describing the maximal P-stable sequence set by n,c1,c2. Furthermore we conjecture that similar equivalences occur in cases if Σ also part of the defining inequality.

Role of copper and cerium species in Cu/CeZSM catalysts for direct methane to methanol reaction: Insights of structure–activity relationship

Role of copper and cerium species in Cu/CeZSM catalysts for direct methane to methanol reaction: Insights of structure–activity relationship

Comprehensive study of Biginelli's compounds show antibacterial activity against Vibrio parahaemolyticus of two strains: In vitro and computational approaches.

This study addresses the critical challenges faced by global aquatic industries such as overfishing, habitat destruction, pollution, climate change, and unsustainable aquaculture practices. It focuses on developing effective solutions by synthesizing potent inhibitors against Vibrio parahaemolyticus of two strains namely: MTCC-451 (A) and Vp-S14 (B). Biginelli's compounds (B1-4) were identified as promising inhibitors with confirmed antibacterial activity through in silico and in vitro studies. Then virtual screening through ADMET, best correlation with regard to QSAR studies, the docking analysis is used to determine, the ligand B1 would be more favorable comparable with others for conducting bacterial studies and DFT calculations show that the optimized structure of ligand B1 has the best FMO values, MEP values and appropriate electronic structural state. This research study is very helpful and matches for "Antibacterial analysis". A total of 99 Biginelli compounds were selected for virtual screening including 2D-QSAR, ADME/T, molecular docking, and DFT calculations. The virtual screened compounds were synthesized then for biological studies. The four highlighted compounds (B1-4) with favorable ADME properties and strong binding affinities compared to gentamicin in both ADME and docking analysis respectively. Further analysis via DFT provided structural insights and active site identification. In vitro assays against pathogenic Vp strains demonstrated significant bactericidal activity, with MIC values of 1.25mg/ml (MTCC-451) and 1mg/ml (Vp-S14).

Nucleotide analogues and mpox: Repurposing the repurposable.

While the COVID-19 crisis is still ongoing, a new public health threat has emerged with recent outbreaks of monkeypox (mpox) infections in Africa. Mass vaccination is not currently recommended by the World Health Organization (WHO), and antiviral treatments are yet to be specifically approved for mpox, although existing FDA-approved drugs (Tecovirimat, Brincidofovir, and Cidofovir) may be used in severe cases or for immunocompromised patients. A first-line of defense is thus drug repurposing, which was heavily attempted against SARS-CoV-2 - albeit with limited success. This review focuses on nucleoside analogues as promising antiviral candidates for targeting of the viral DNA-dependent DNA polymerase. In contrast to broad-spectrum screening approaches employed for SARS-CoV-2, we emphasize the importance of understanding the structural specificity of viral polymerases for rational selection of potential candidates. By comparing DNA-dependent DNA polymerases with other viral polymerases, we highlight the unique features that influence the efficacy and selectivity of nucleoside analogues. These structural insights provide a framework for the preselection, repurposing, optimization, and design of nucleoside analogues, aiming to accelerate the development of targeted antiviral therapies for mpox and other viral infections.

How Do the Valency and Radii of Cations Affect the Rheological Properties of Aqueous Solutions of Zwitterionic and Anionic Surfactant Mixtures?

Despite extensive research on the use of salts to enhance micellar growth, numerous questions remain regarding the impact of ionic exchange and molecular structure on charge neutralization. This study looks into how certain cations (Na+, Ca2+, and Mg2+) affect the structure of a cocamidopropyl betaine CAPB and sodium dodecylbenzenesulfonate SDBS surfactant mixture, aiming toward applications in targeted delivery systems. The mixture consists of a zwitterionic surfactant, cocamidopropyl betaine (CAPB), and an anionic surfactant, sodium dodecylbenzenesulfonate (SDBS), combined in varying molar ratios at a total concentration of 200 mM. We characterized the macroscale properties through rheological measurements and obtained detailed structural insights using small-angle X-ray scattering (SAXS) and cryogenic electron microscopy (cryo-EM). The findings reveal that increasing the concentration of cations in the CAPB/SDBS mixture induces the formation of peaks in the zero-shear viscosity as a function of salt concentration (salt curve). Analysis through cryo-EM and SAXS showed that these viscosity peaks are related to the change of micellar assemblies from entangled worm-like micelles to branched worm-like micelles and then to bilayer structures (vesicles). The specific cation concentration at which the zero-shear viscosity peak occurs, as well as the maximum viscosity, is strongly influenced by the type of cation present in the CAPB/SDBS solutions, a phenomenon explained by the Hofmeister series. Notably, the differing affinities of cations for the carboxylate COO- and sulfite SO3- groups and the partial dehydration of micelles contribute to the lower concentration of magnesium cations required to reach the viscosity peak compared to calcium cations.

Structural insights into the role of reduced cysteine residues in SOD1 amyloid filament formation

The formation of superoxide dismutase 1 (SOD1) filaments has been implicated in amyotrophic lateral sclerosis (ALS). Although the disulfide bond formed between Cys57 and Cys146 in the active state has been well studied, the role of the reduced cysteine residues, Cys6 and Cys111, in SOD1 filament formation remains unclear. In this study, we investigated the role of reduced cysteine residues by determining and comparing cryoelectron microscopy (cryo-EM) structures of wild-type (WT) and C6A/C111A SOD1 filaments under thiol-based reducing and metal-depriving conditions, starting with protein samples possessing enzymatic activity. The C6A/C111A mutant SOD1 formed filaments more rapidly than the WT protein. The mutant structure had a unique paired-protofilament arrangement, with a smaller filament core than that of the single-protofilament structure observed in WT SOD1. Although the single-protofilament form developed more slowly, cross-seeding experiments demonstrated the predominance of single-protofilament morphology over paired protofilaments, regardless of the presence of the Cys6 and Cys111 mutations. These findings highlight the importance of the number of amino acid residues within the filament core in determining the energy requirements for assembly. Our study provides insights into ALS pathogenesis by elucidating the initiation and propagation of filament formation, which potentially leads to deleterious amyloid filaments.

Isolation, identification, and characterisation of the malachite green detoxifying bacterial strain Bacillus pacificus ROC1 and the azoreductase AzrC

Malachite green (MG) is used as a dye for materials such as wood, cotton, and nylon, and is used in aquaculture to prevent fungal and protozoan diseases. However, it is highly toxic, with carcinogenic, mutagenic, and teratogenic properties, resulting in bans worldwide. Despite this, MG is still frequently used in many countries due to its efficacy and economy. MG is persistent in the environment and so requires degradative intervention. In this work we isolated Bacillus pacificus ROC1 strain from a salt flat in Pakistan that had the ability to aerobically detoxify MG, as determined by bacterio- and phyto-toxicity assays. We demonstrate immobilized B. pacificus ROC1 can effectively detoxify MG, which highlights a potential method for its biodegradation. Genomic sequencing identified three candidate azo-reductases within B. pacificus ROC1 that could be responsible for the MG-degrading activity. These were cloned, expressed and purified from Escherichia coli, with one (AzrC), catalyzing the reduction of MG to leuco-MG in vitro. AzrC was crystallised and MG was captured within the active site in a Michaelis complex, providing structural insight into the reduction mechanism. Altogether, this work identifies a bacterium capable of aerobically degrading a major industrial pollutant and characterizes the molecular basis for this activity.

Structural and evolutionary insights into the functioning of glycoprotein hormones and their receptors.

The neuroendocrine system that comprises the glycoprotein hormones (GpHs) and their receptors is essential for reproduction and metabolism. Each GpH hormone is an αβ heterodimer of cystine-knot proteins and its cognate receptor is a G-protein coupled receptor (GPCR) distinguished by a large leucine-rich-repeat (LRR) extracellular domain that binds the hormone and a class A GPCR transmembrane domain that signals through an associating heterotrimeric G protein. Hence, the receptors are called LRR-containing GPCRs-LGRs. The vertebrate GpHs and LGRs have co-evolved from homologs in the earliest metazoan animals, including sponges and comb jellies, but these are absent from unicellular organisms and plants. The two GpH subunits and accompanying LGR receptor of the nematode Caenorhabditis elegans are representative of the invertebrate evolutionary predecessors of human GpH proteins and their receptors, for example follicle-stimulating hormone (FSH) and the FSH receptor (FSHR). Atomic structures of the human GpHs and their receptors, which have been determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM), inform the evolutionary process and provide a mechanistic understanding of the transmission of biochemical signals of hormone binding at the cell surface to the elicitation of second messengers such as cyclic AMP in the cytoplasm. There is compelling biochemical and cellular evidence for the importance of receptor dimers in GpH signaling in cells; yet, all of the human receptors are monomeric as defined beautifully by cryo-EM. Fortunately, the LGR of C. elegans is a stable dimer and its structure, when analyzed in the context of structural information from the human counterparts, predicts a hypothetical model for functionally relevant dimeric associations of the human GpH receptors.

Structural insights into HIV-2 CA lattice formation and FG-pocket binding revealed by single-particle cryo-EM.

One of the striking features of human immunodeficiency virus (HIV) is the capsid, a fullerene cone comprised of pleomorphic capsid protein (CA) that shields the viral genome and recruits cofactors. Despite significant advances in understanding the mechanisms of HIV-1 CA assembly and host factor interactions, HIV-2 CA assembly remains poorly understood. By templating the assembly of HIV-2 CA on functionalized liposomes, we report high-resolution structures of the HIV-2 CA lattice, including both CA hexamers and pentamers, alone and with peptides of host phenylalanine-glycine (FG)-motif proteins Nup153 and CPSF6. While the overall fold and mode of FG-peptide binding is conserved with HIV-1, this study reveals distinctive features of the HIV-2 CA lattice, including differing structural character at regions of host factor interactions and divergence in the mechanism of formation of CA hexamers and pentamers. This study extends our understanding of HIV capsids and highlights an approach facilitating the study of lentiviral capsid biology.

Enhanced Charge Transport through Ion Networks in Highly Concentrated LiSCN‐Polyethylene Carbonate Solid Polymer Electrolytes

Challenging the preference for bulky anions due to low binding energy with Li+ ion, the lithium thiocyanate‐polyethylene carbonate (LiSCN‐PEC) solid polymer electrolyte (SPE) demonstrates higher ionic conductivities (3.16 × 10−5 S cm−1) at polymer‐in‐salt concentration (100 mol%) compared to those with lithium bis(fluorosulfonyl)imide (LiFSI, 1.01 × 10−5 S cm−1) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, 1.72 × 10−7 S cm−1). Through the careful selection of PEC and LiSCN as components of SPE, the carbonyl stretching of PEC and the SCN− stretching band as vibrational reporters provide detailed structural insights into the Li+ ion transport channel. Spectroscopic investigations reveal that enhanced ion aggregation alters the solvation structure around the Li+ and diminishes the interaction between Li+ and polymer (PEC) with increasing LiSCN concentrations, promoting faster segmental motion as a major transport mechanism. However, the transition observed from subionic to superionic behavior in the Walden plot indicates the onset of segmental motion decoupled charge transport pathway. The SCN− vibrational spectrum elucidates the evolution from a Li–SCN–Li type chain‐like structure to a Li2 > SCN < Li2 type extended ion network with increasing LiSCN concentration, revealing that the ion network provides an alternative channel for Li+ ion transfer at higher concentrations, enhancing conductivity.

Kinetic and Structural Insights into β-Cyclodextrin Complexation with Asparagine Enantiomers: An Experimental and Theoretical Study

We report on the dynamic interactions between β-cyclodextrin (β-CD) and each one of the two enantiomers of asparagine (d-Asp, l-Asp). Molecular docking methodologies were applied to elucidate the formation of the β-CD—d-Asp and β-CD—l-Asp inclusion complexes. Ultrasonic relaxation spectra revealed a single relaxation process in the frequency range studied that is attributed to the complexation between β-CD and asparagine enantiomers. Kinetic parameters and thermodynamic properties for each system were determined directly from the concentration- and temperature-dependent acoustic measurements, respectively. Both β-CD—d-Asp and β-CD—l-Asp systems revealed subtle differences in their thermodynamic and kinetic properties. The infrared absorption spectra of the host molecule, the guest enantiomers, and both inclusion complexes were recorded to verify and further elucidate the complexation mechanism. DFT methodologies were performed to calculate the theoretical IR spectra of the inclusion complexes and compared with the corresponding experimental spectra. The close resemblance between the experimental and theoretically predicted IR spectra is supportive of the formation of inclusion complexes. The encapsulation of asparagine enantiomers in β-cyclodextrin enables not only applications in drug delivery but also the detection and separation of chimeric molecules.

Structural model of a bacterial focal adhesion complex

Cell movement on surfaces relies on focal adhesion complexes (FAs), which connect cytoskeletal motors to the extracellular matrix to produce traction forces. The soil bacterium Myxococcus xanthus uses a bacterial FA (bFA), for surface movement and predation. The bFA system, known as Agl-Glt, is a complex network of at least 17 proteins spanning the cell envelope. Despite understanding the system dynamics, its molecular structure and protein interactions remain unclear. In this study, we utilize AlphaFold to generate models based on the known interactions and dynamics of gliding motility proteins. This approach provides us with a comprehensive view of the interactions across the entire complex. Our structural insights show the connection of essential functional modules throughout the cell envelope and offer an inspiring view of the force transduction mechanism from the inner molecular motor to the exterior of the cell.

Pharmacological and structural insights into nanvuranlat, a selective LAT1 (SLC7A5) inhibitor, and its N-acetyl metabolite with implications for cancer therapy

L-type amino acid transporter 1 (LAT1, SLC7A5), overexpressed in various cancers, mediates the uptake of essential amino acids crucial for tumor growth. It has emerged as a promising target for cancer therapy. Nanvuranlat (JPH203/KYT-0353), a LAT1 inhibitor, has shown antitumor activity in preclinical studies and efficacy in biliary tract cancer during clinical trials. This study provides a comprehensive pharmacological characterization of nanvuranlat and its N-acetyl metabolite, including structural insights into their LAT1 interactions. Both compounds demonstrated high selectivity for LAT1 over LAT2 and other amino acid transporters. Nanvuranlat acts as a competitive, non-transportable LAT1 inhibitor (Ki = 38.7 nM), while its N-acetyl metabolite retains selectivity but with reduced affinity (Ki = 1.68 µM). Nanvuranlat exhibited a sustained inhibitory effect on LAT1 even after its removal, indicating the potential for prolonged therapeutic effects. Both compounds showed comparable dissociation rates, suggesting that N-acetylation does not affect the interaction responsible for slow dissociation. The U-shaped conformation adopted by nanvuranlat when bound to LAT1 likely contributes to its high affinity, selectivity, sustained inhibitory effect, and non-transportable nature observed in this study. These insights into nanvuranlat’s mechanism and metabolic impact provide essential information for understanding its clinical efficacy and advancing LAT1-targeted cancer therapies.

Structural Insights into Broad-Range Polyphosphate Kinase2‑II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis.

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP‑consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85% of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49%. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

Structural insights into glucose-6-phosphate recognition and hydrolysis by human G6PC1

The glucose-6-phosphatase (G6Pase) is an integral membrane protein that catalyzes the hydrolysis of glucose-6-phosphate (G6P) in the endoplasmic reticulum lumen and plays a vital role in glucose homeostasis. Dysregulation or genetic mutations of G6Pase are associated with diabetes and glycogen storage disease 1a (GSD-1a). Studies have characterized the biophysical and biochemical properties of G6Pase; however, the structure and substrate recognition mechanism of G6Pase remain unclear. Here, we present two cryo-EM structures of the 40-kDa human G6Pase: a wild-type apo form and a mutant G6Pase-H176A with G6P bound, elucidating the structural basis for substrate recognition and hydrolysis. G6Pase comprises nine transmembrane helices and possesses a large catalytic pocket facing the lumen. Unexpectedly, G6P binding induces substantial conformational rearrangements in the catalytic pocket, which facilitate the binding of the sugar moiety. In conjunction with functional analyses, this study provides critical insights into the structure, substrate recognition, catalytic mechanism, and pathology of G6Pase.