Finding the sweet spot of the insect gustatory receptor.

In a recent issue of Nature, Gomes et al.1 utilized structural, experimental, and computational biology to investigate the ligand-gated activation of BmGr9, an insect gustatory receptor specifically tuned to D-fructose. Together with two other studies published elsewhere, they are the first to describe how sugars bind to insect gustatory receptors.

Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

Structure-based design of a Plasmodium vivax Duffy-binding protein immunogen focuses the antibody response to functional epitopes.

The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development.

Probing scaffold size effects on multivalent lectin-glycan binding affinity, thermodynamics and antiviral properties using polyvalent glycan-gold nanoparticles.

Multivalent lectin-glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for understanding their basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remains poorly understood, greatly limiting research progress. We have recently developed densely glycosylated nanoparticles, e.g., ∼4 nm quantum dots (QDs) or ∼5 nm gold nanoparticles (GNPs), as mechanistic probes for MLGIs. Using two important model lectin viral receptors, DC-SIGN and DC-SIGNR, we have shown that these probes can not only offer sensitive fluorescence assays for quantifying MLGI affinities, but also reveal key structural information (e.g., binding site orientation and binding mode) useful for MLGI targeting. However, the small sizes of the previous scaffolds may not be optimal for maximising MLGI affinity and targeting specificity. Herein, using α-manno-α-1,2-biose (DiMan) functionalised GNP (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format to minimise the possible interference of GNP's strong inner filter effect in MLGI affinity quantification, revealing that increasing the GNP size is highly beneficial for enhancing MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity and Van't Hoff analyses, revealing that GNP-DiMan-DC-SIGN/R binding is enthalpy driven with favourable binding Gibbs free energy changes (ΔG°) being enhanced with increasing GNP size. Finally, we show that increasing the GNP size significantly enhances their antiviral potency. Notably, the DiMan coated 27 nm GNP potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC50 of ∼23 and ∼49 pM, respectively, making it the most potent glycoconjugate inhibitor against DC-SIGN/R-mediated Ebola cellular infections. Our results have established GNP-glycans as a new tool for quantifying MLGI biophysical parameters and revealed that increasing the GNP scaffold size significantly enhances their MLGI affinities and antiviral potencies.

Sugar detection in 3D: Structure of an insect gustatory receptor.

The insect gustatory receptors (Grs) are one of the largest families of ion channels in the animal kingdom. Frank et al.1 unveil the structure of a fructose-sensing Gr and provide insight into its function.

Thermodynamics-Guided Design Reveals a Cooperative Hydrogen Bond in DC-SIGN-targeted Glycomimetics.

Due to the shallow and hydrophilic binding sites of carbohydrate-binding proteins, the design of glycomimetics is often complicated by high desolvation costs as well as competition with solvent. Therefore, a careful optimization of interaction vectors and ligand properties is required in the design and optimization of glycomimetics. Here, we employ thermodynamics-guided design to optimize mannose-based glycomimetics targeting the human C-type lectin receptor dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN), a pathogenic host factor in viral infections. By exploring ligand rigidification and hydrogen bond engineering, a monovalent glycomimetic with an unprecedented affinity for DC-SIGN in the low µM range was discovered. A matched molecular pair analysis based on microcalorimetric data revealed a stereospecific hydrogen bond interaction with Glu358/Ser360 as the origin of this cooperative and enthalpically dominated interaction. This detailed insight into the binding mechanism paves the way for an improvement of monovalent glycomimetics targeting DC-SIGN.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

Binding Evolution of the Dengue Virus Envelope Against DC-SIGN: A Combined Approach of Phylogenetics and Molecular Dynamics Analyses Over 30 Years of Dengue Virus in Brazil.

The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.

GMMA Can Stabilize Proteins Across Different Functional Constraints.

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.

Structural basis for the immune recognition and selectivity of the immune receptor PVRIG for ligand Nectin-2.

Nectin and nectin-like (Necl) co-receptor axis, comprised of receptors DNAM-1, TIGIT, CD96, PVRIG, and nectin/Necl ligands, is gaining prominence in immuno-oncology. Within this axis, the inhibitory receptor PVRIG recognizes Nectin-2 with high affinity, but the underlying molecular basis remains unknown. By determining the crystal structure of PVRIG in complex with Nectin-2, we identified a unique CC' loop in PVRIG, which complements the double-lock-and-key binding mode and contributes to its high affinity for Nectin-2. The association of the corresponding charged residues in the F-strands explains the ligand selectivity of PVRIG toward Nectin-2 but not for Necl-5. Moreover, comprehensive comparisons of the binding capacities between co-receptors and ligands provide innovative insights into the intra-axis immunoregulatory mechanism. Taken together, these findings broaden our understanding of immune recognition and regulation mediated by nectin/Necl co-receptors and provide a rationale for the development of immunotherapeutic strategies targeting the nectin/Necl axis.

Secondary Sites of the C-type Lectin-Like Fold.

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: 2. The Role of Symmetry in GTPase Binding and Dimer Formation.

The Rho GTPase binding domain of Plexin-B1 (RBD) prevails in solution as dimer. Under appropriate circumstances, it binds the small GTPase Rac1 to yield the complex RBD-Rac1. Here, we study RBD dimerization and complex formation from a symmetry-based perspective using data derived from 1 µs long MD simulations. The quantities investigated are the local potentials, u(MD), prevailing at the N-H sites of the protein. These potentials are statistical in character providing an empirical description of the local structure. To establish more methodical description, a method for approximating them by explicit functions, u(simulated), was developed in the preceding article in this journal issue. These functions are combinations of analytical Wigner functions, DL,K, belonging to the D2h point group. The D2h subgroups Ag and B2u are found to dominate u(simulated); the B1u subgroup contributes in some cases. The Ag (B2u) functions have axial or rhombic symmetry. For the first time, local potentials in proteins can be quantitatively characterized in terms of their strength (rhombicity) evaluated by axial Ag (rhombic Ag and B2u) contributions. Until now, the chain-segment [ß3-L3-ß4] and to some extent the α2-helix have been associated with GTPase binding. Here, we find that this process causes an increase (decrease) in the potential strength of ß3 and ß4 (the preceding L2 loop and the remote chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting effects of counterbalancing and allostery. There is evidence for the L2 loop being associated with RBD-GTPase binding. Until now only the L4 loop has been associated with RBD dimerization. The latter process is found to cause an increase (decrease) in the potential strength and rhombicity of the L4 loop (the adjacent chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting counterbalancing activity. On average, the RBD dimer features stronger local potentials than RBD-Rac1. The novel information inherent in these findings is mesoscopic in character. Prospects of interest include exploring relation to atomistic force-field parameters.

Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli.

This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections.

Crystal structures of human immune protein FIBCD1 suggest an extended binding site compatible with recognition of pathogen-associated carbohydrate motifs.

Fibrinogen C domain-containing protein 1 (FIBCD1) is an immune protein proposed to be involved in host recognition of chitin on the surface of pathogens. As FIBCD1 readily binds acetylated molecules, we have determined the high-resolution crystal structures of a recombinant fragment of the FIBCD1 C-terminal domain complexed with small N-acetyl-containing ligands to determine the mode of recognition. All ligands bind at the conserved N-acetyl-binding site (S1) with galactose and glucose-derived ligands rotated 180° relative to each other. One subunit of a native structure derived from protein expressed in mammalian cells binds glycosylation from a neighboring subunit, in an extended binding site. Across the various structures, the primary S1 binding pocket is occupied by N-acetyl-containing ligands or acetate, with N-acetyl, acetate, or sulfate ion in an adjacent pocket S1(2). Inhibition binding studies of N-acetylglucosamine oligomers, (GlcNAc)n, n = 1, 2, 3, 5, 11, via ELISA along with microscale thermophoresis affinity assays indicate a strong preference of FIBCD1 for longer N-acetylchitooligosaccharides. Binding studies of mutant H396A, located beyond the S1(2) site, showed no significant difference from wildtype, but K381L, within the S1(2) pocket, blocked binding to the model ligand acetylated bovine serum albumin, suggesting that S1(2) may have functional importance in ligand binding. The binding studies, alongside structural definition of diverse N-acetyl monosaccharide binding in the primary S1 pocket and of additional, adjacent binding pockets, able to accommodate both carbohydrate and sulfate functional groups, suggest a versatility in FIBCD1 to recognize chitin oligomers and other pathogen-associated carbohydrate motifs across an extended surface.

The molecular structure and function of fibrocystin, the key gene product implicated in autosomal recessive polycystic kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease is an early onset inherited hepatorenal disorder affecting around 1 in 20,000 births with no approved specific therapies. The disease is almost always caused by variations in the polycystic kidney and hepatic disease 1 gene, which encodes fibrocystin (FC), a very large, single-pass transmembrane glycoprotein found in primary cilia, urine and urinary exosomes. By comparison to proteins involved in autosomal dominant PKD, our structural and molecular understanding of FC has lagged far behind such that there are no published experimentally determined structures of any part of the protein. Bioinformatics analyses predict that the ectodomain contains a long chain of immunoglobulin-like plexin-transcription factor domains, a protective antigen 14 domain, a tandem G8-TMEM2 homology region and a sperm protein, enterokinase and agrin domain. Here we review current knowledge on the molecular function of the protein from a structural perspective.

The (Pro)renin Receptor - A Regulatory Nodal Point in Disease Networks.

Experimental inhibition of the (pro)renin receptor [(P)RR] is a promising therapeutic strategy in different disease models ranging from cardiorenal to oncological entities. Here, we briefly review the direct protein-protein interaction partners of the (P)RR and the plethora of distinct diseases in which the (P)RR is involved. The first structural work on the (P)RR using AlphaFold, which was recently published by Ebihara et al., is the center of this mini-review since it can mechanistically link the protein-protein interaction level with the pathophysiological level. More detailed insights into the 3D structure of the (P)RR and its interaction domains might guide drug discovery on this novel target. Finally, antibody- and small molecule-based approaches to inhibit the (P)RR are shortly discussed.

Validation of agent-based models of surface receptor oligomerisation.

Receptor dimerisation and higher order oligomerisation regulates signalling by a wide variety of transmembrane receptors. We discuss how agent-based modelling (ABM) combined with advanced microscopy and structural studies can provide new insights into the regulation of clustering, including spatial considerations, revealing novel targets for therapeutic intervention.

A disulfide chaperone knockout facilitates spin labeling and pulse EPR spectroscopy of outer membrane transporters.

Pulse EPR measurements provide information on distances and distance distributions in proteins but require the incorporation of pairs of spin labels that are usually attached to engineered cysteine residues. In previous work, we demonstrated that efficient in vivo labeling of the Escherichia coli outer membrane vitamin B12 transporter, BtuB, could only be achieved using strains defective in the periplasmic disulfide bond formation (Dsb) system. Here, we extend these in vivo measurements to FecA, the E. coli ferric citrate transporter. As seen for BtuB, pairs of cysteines cannot be labeled when the protein is present in a standard expression strain. However, incorporating plasmids that permit an arabinose induced expression of FecA into a strain defective in the thiol disulfide oxidoreductase, DsbA, enables efficient spin-labeling and pulse EPR of FecA in cells. A comparison of the measurements made on FecA in cells with measurements made in reconstituted phospholipid bilayers suggests that the cellular environment alters the behavior of the extracellular loops of FecA. In addition to these in situ EPR measurements, the use of a DsbA minus strain for the expression of BtuB improves the EPR signals and pulse EPR data obtained in vitro from BtuB that is labeled, purified, and reconstituted into phospholipid bilayers. The in vitro data also indicate the presence of intermolecular BtuB-BtuB interactions, which had not previously been observed in a reconstituted bilayer system. This result suggests that in vitro EPR measurements on other outer membrane proteins would benefit from protein expression in a DsbA minus strain.

Discovery of Potent Tetrazole Free Fatty Acid Receptor 2 Antagonists.

The free fatty acid receptor 2 (FFA2), also known as GPR43, mediates effects of short-chain fatty acids and has attracted interest as a potential target for treatment of various metabolic and inflammatory diseases. Herein, we report the results from bioisosteric replacement of the carboxylic acid group of the established FFA2 antagonist CATPB and SAR investigations around these compounds, leading to the discovery of the first high-potency FFA2 antagonists, with the preferred compound TUG-2304 (16l) featuring IC50 values of 3-4 nM in both cAMP and GTPγS assays, favorable physicochemical and pharmacokinetic properties, and the ability to completely inhibit propionate-induced neutrophil migration and respiratory burst.

Organometallic anti-tumor agents: targeting from biomolecules to dynamic bioprocesses.

The great clinical success of cisplatin and its derivatives has convinced people that metal complexes could play a more significant role in human cancer therapy. However, targeting and drug resistance are still two dominant problems that need to be urgently solved for metallodrugs' efficacy and clinical translation. As an important component of metal complexes, organometallics have been experiencing rapid development in recent years. Compared with platinum drugs, emerging anti-tumor organometallics targeting dynamic bioprocesses provide an effective strategy to overcome conventional problems. This review focuses on burgeoning anti-tumor strategies and provides up-to-date advances in anti-tumor organometallics development based on their action mechanisms. Specifically, important tumor-overexpressed proteins and nucleic acids as organometallics' anti-tumor targets are systematically presented, followed by organometallics that exert their anti-tumor activity by perturbing tumor intracellular energy/redox/metal/immune homeostasis. Finally, nine cell death pathways including apoptosis, paraptosis, autophagy, oncosis, necrosis, necroptosis, ferroptosis, pyroptosis, and immunogenic cell death (ICD) that can be induced by organometallics are reviewed, and their morphological and biochemical features are summarised. This review at the interface of chemistry, biology, and medicine aims to enlighten the rational development of organometallic anti-tumor agents.