Reversible dougong structured receptor-ligand recognition for building dynamic extracellular matrix mimics.

Dynamic biomaterials excel at recapitulating the reversible interlocking and remoldable structure of the extracellular matrix (ECM), particularly in manipulating cell behaviors and adapting to tissue morphogenesis. While strategies based on dynamic chemistries have been extensively studied for ECM-mimicking dynamic biomaterials, biocompatible molecular means with biogenicity are still rare. Here, we report a nature-derived strategy for fabrication of dynamic biointerface as well as a three-dimensional (3D) hydrogel structure based on reversible receptor-ligand interaction between the glycopeptide antibiotic vancomycin and dipeptide d-Ala-d-Ala. We demonstrate the reversible regulation of multiple cell types with the dynamic biointerface and successfully implement the dynamic hydrogel as a functional antibacterial 3D scaffold to treat tissue repair. In view of the biogenicity and high applicability, this nature-derived reversible molecular strategy will bring opportunities for malleable biomaterial design with great potential in biomedicine.

Binding characteristics of the doxepin E/Z-isomers to the histamine H1 receptor revealed by receptor-bound ligand analysis and molecular dynamics study.

Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H1 receptor (H1R) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the H1R/doxepin complex, the hydroxyl group of Thr1123.37 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer. The detailed binding characteristics and reasons for the differences remain unclear. In this study, we analyzed doxepin isomers bound to the receptor following extraction from a purified H1R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H1R was 55:45, indicating that the Z-isomer was bound to WT H1R with an approximately 5.2-fold higher affinity than the E-isomer. For the T1123.37V mutant, the E/Z ratio was 89:11, indicating that both isomers have similar affinities. Free energy calculations using molecular dynamics (MD) simulations also reproduced the experimental results of the relative binding free energy differences between the isomers for WT and T1123.37V. Furthermore, MD simulations revealed that the hydroxyl group of T1123.37 did not form hydrogen bonds with the E-isomer, but with the adjacent residues in the binding pocket. Analysis of the receptor-bound doxepin and MD simulations suggested that the hydroxyl group of T1123.37 contributes to the formation of a chemical environment in the binding pocket, which is slightly more favorable for the Z-isomer without hydrogen bonding with doxepin.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors.

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

A membrane protein display platform for receptor interactome discovery.

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor-ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface-expressed target of interest in its native state.

N-Glycolylneuraminic Acid Binding of Avian and Equine H7 Influenza A Viruses.

Influenza A viruses (IAV) initiate infection by binding to glycans with terminal sialic acids on the cell surface. Hosts of IAV variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in most mammals, including horses and pigs, but is absent in humans, ferrets, and birds. The only known naturally occurring IAV that exclusively bind NeuGc are extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with NeuGc and observed high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs and an H15 HA. We found that the A135E mutation is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. The additional mutations S128T, I130V, T189A, and K193R converted the specificity from NeuAc to NeuGc. We investigated the residues at positions 128, 130, 135, 189, and 193 in a phylogenetic analysis of avian and equine H7 HAs. This analysis revealed a clear distinction between equine and avian residues. The highest variability was observed at key position 135, of which only the equine glutamic acid led to NeuGc binding. These results demonstrate that genetically distinct H7 and H15 HAs can be switched from NeuAc to NeuGc binding and vice versa after the introduction of several mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. IMPORTANCE Influenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A viruses, these "keys" (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority bind NeuGc. NeuGc is present in species like horses, pigs, and mice but not in humans, ferrets, and birds. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

Synthesis, molecular docking, and binding Gibbs free energy calculation of ß-nitrostyrene derivatives: Potential inhibitors of SARS-CoV-2 3CL protease.

The outbreak of novel coronavirus disease 2019 (COVID-19), caused by the novel coronavirus (SARS-CoV-2), has had a significant impact on human health and the economic development. SARS-CoV-2 3CL protease (3CLpro) is highly conserved and plays a key role in mediating the transcription of virus replication. It is an ideal target for the design and screening of anti-coronavirus drugs. In this work, seven ß-nitrostyrene derivatives were synthesized by Henry reaction and ß-dehydration reaction, and their inhibitory effects on SARS-CoV-2 3CL protease were identified by enzyme activity inhibition assay in vitro. Among them, 4-nitro-ß-nitrostyrene (compound a) showed the lowest IC50 values of 0.7297 µM. To investigate the key groups that determine the activity of ß-nitrostyrene derivatives and their interaction mode with the receptor, the molecular docking using the CDOCKER protocol in Discovery Studio 2016 was performed. The results showed that the hydrogen bonds between ß-NO2 and receptor GLY-143 and the π-π stacking between the aryl ring of the ligand and the imidazole ring of receptor HIS-41 significantly contributed to the ligand activity. Furthermore, the ligand-receptor absolute binding Gibbs free energies were calculated using the Binding Affinity Tool (BAT.py) to verify its correlation with the activity of ß-nitrostyrene 3CLpro inhibitors as a scoring function. The higher correlation(r2=0.6) indicates that the absolute binding Gibbs free energy based on molecular dynamics can be used to predict the activity of new ß-nitrostyrene 3CLpro inhibitors. These results provide valuable insights for the functional group-based design, structure optimization and the discovery of high accuracy activity prediction means of anti-COVID-19 lead compounds.

Repurposing of World-Approved Drugs for Potential Inhibition against Human Carbonic Anhydrase I: A Computational Study.

Human carbonic anhydrases (hCAs) have enzymatic activities for reversible hydration of CO2 and are acknowledged as promising targets for the treatment of various diseases. Using molecular docking and molecular dynamics simulation approaches, we hit three compounds of methyl 4-chloranyl-2-(phenylsulfonyl)-5-sulfamoyl-benzoate (84Z for short), cyclothiazide, and 2,3,5,6-tetrafluoro-4-piperidin-1-ylbenzenesulfonamide (3UG for short) from the existing hCA I inhibitors and word-approved drugs. As a Zn2+-dependent metallo-enzyme, the influence of Zn2+ ion models on the stability of metal-binding sites during MD simulations was addressed as well. MM-PBSA analysis predicted a strong binding affinity of -18, -16, and -14 kcal/mol, respectively, for these compounds, and identified key protein residues for binding. The sulfonamide moiety bound to the Zn2+ ion appeared as an essential component of hCA I inhibitors. Vina software predicted a relatively large (unreasonable) Zn2+-sulfonamide distance, although the relative binding strength was reproduced with good accuracy. The selected compounds displayed potent inhibition against other hCA isoforms of II, XIII, and XIV. This work is valuable for molecular modeling of hCAs and further design of potent inhibitors.

Moderate Constraint Facilitates Association and Force-Dependent Dissociation of HA-CD44 Complex.

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction of CD44 HABD (HA binding site domain) to HA through free and steered molecular dynamics (MD) simulations as well as atomic force microscope (AFM) measurement using different constraints on HA. The middle, two ends or both of the constrained HA chains were fixed for MD simulations, while one and two biotin-avidin linkage or physical absorption were used to immobilize HA on substrates for AFM experiments, to model HA chains with low, moderate and high HA flexibilities, respectively. We found that binding of CD44 to moderate fixed HA was possessed of a better thermo-stability, a lower mechanical strength and a higher dissociation probability, while higher adhesive frequency, smaller rupture force and shorter lifetime were assigned to CD44 on the two biotin-immobilized HA rather than one biotin-immobilized or physically absorbed HA on substrates, suggesting a moderate HA flexibility requirement in favor of association and force-induced dissociation of CD44-HA complex. Tensile-induced convex conformation of HA chain was responsible for reduction of complex mechano-stability and did inversely a shrunken CD44 HABD under stretching; transition from catch bond to slip bond governed CD44-HA interaction. This study uncovered the regulation mechanism and its molecular basis for CD44-HA affinity under diverse mechano-microenvironments and provided a new insight into CD44-HA interaction-mediated cell inflammatory responses and tumor development.

Study of the Sensing Kinetics of G Protein-Coupled Estrogen Receptor Sensors for Common Estrogens and Estrogen Analogs.

Endogenous and exogenous estrogens are widely present in food and food packaging, and high levels of natural estrogens and the misuse or illegal use of synthetic estrogens can lead to endocrine disorders and even cancer in humans. Therefore, it is consequently important to accurately evaluate the presence of food-functional ingredients or toxins with estrogen-like effects. In this study, an electrochemical sensor based on G protein-coupled estrogen receptors (GPERs) was fabricated by self-assembly, modified by double-layered gold nanoparticles, and used to measure the sensing kinetics for five GPER ligands. The interconnected allosteric constants (Ka) of the sensor for 17ß-estradiol, resveratrol, G-1, G-15, and bisphenol A were 8.90 × 10-17, 8.35 × 10-16, 8.00 × 10-15, 5.01 × 10-15, and 6.65 × 10-16 mol/L, respectively. The sensitivity of the sensor for the five ligands followed the order of 17ß-estradiol > bisphenol A > resveratrol > G-15 > G-1. The receptor sensor also demonstrated higher sensor sensitivity for natural estrogens than exogenous estrogens. The results of molecular simulation docking showed that the residues Arg, Glu, His, and Asn of GPER mainly formed hydrogen bonds with -OH, C-O-C, or -NH-. In this study, simulating the intracellular receptor signaling cascade with an electrochemical signal amplification system enabled us to directly measure GPER-ligand interactions and explore the kinetics after the self-assembly of GPERs on a biosensor. This study also provides a novel platform for the accurate functional evaluation of food-functional components and toxins.

Coreceptor functions of cell surface heparan sulfate proteoglycans.

Receptor-ligand interactions play an important role in many biological processes by triggering specific cellular responses. These interactions are frequently regulated by coreceptors that facilitate, alter, or inhibit signaling. Coreceptors work in parallel with other specific and accessory molecules to coordinate receptor-ligand interactions. Cell surface heparan sulfate proteoglycans (HSPGs) function as unique coreceptors because they can bind to many ligands and receptors through their HS and core protein motifs. Cell surface HSPGs are typically expressed in abundance of the signaling receptors and, thus, are capable of mediating the initial binding of ligands to the cell surface. HSPG coreceptors do not possess kinase domains or intrinsic enzyme activities and, for the most part, binding to cell surface HSPGs does not directly stimulate intracellular signaling. Because of these features, cell surface HSPGs primarily function as coreceptors for many receptor-ligand interactions. Given that cell surface HSPGs are widely conserved, they likely serve fundamental functions to preserve basic physiological processes. Indeed, cell surface HSPGs can support specific cellular interactions with growth factors, morphogens, chemokines, extracellular matrix (ECM) components, and microbial pathogens and their secreted virulence factors. Through these interactions, HSPG coreceptors regulate cell adhesion, proliferation, migration, and differentiation, and impact the onset, progression, and outcome of pathophysiological processes, such as development, tissue repair, inflammation, infection, and tumorigenesis. This review seeks to provide an overview of the various mechanisms of how cell surface HSPGs function as coreceptors.

T helper cells expressing fractalkine receptor and bearing T follicular helper 1-like cell functions in patients with IgG4-related disease.

OBJECTIVE: Because the pathological features of IgG4-related disease (IgG4-RD) include lymphocyte infiltration and fibrotic changes in the lesions, we investigated the significance of fractalkine (CX3CL1) and lymphocyte subsets in patients with IgG4-RD. METHODS: Peripheral blood and biopsied samples were obtained from healthy controls (HCs, n = 10), RA (n = 10) and IgG4-RD patients (n = 16) and were analysed by flow cytometry, immunohistology and costimulation assays. RESULTS: Peripheral CX3CR1+ CD4+ T cells had an approximately 3-fold increase in the IgG4-RD patients (15.4%), compared with the HCs (5.0%). In addition, CX3CR1+ CD4+ T cells were localized in the salivary glands of the IgG4-RD patients but not in those with Sicca syndrome. CX3CR1 was induced on 20% of CD4+ T cells after T-cell receptor (TCR) simulation with IL-12 for five days culture. CX3CR1+ T cells showed high expression of both CXCR5 and CXCR3. Moreover, they co-expressed Bcl-6 and T-bet, the master transcription factors for T helper 1 (Th1) and T follicular helper (Tfh) cells. After secondary stimulation, CX3CR1+ T cells produced both IFN-gamma (IFN-γ) and IL-21. Compared with their CX3CR1- counterparts, CX3CR1+ CD4+ T cells induced plasmablast differentiation from naïve B cells more efficiently (15.0 vs 5.0%) and increased the production of IgG2, IgG3 and IgG4 by B cells. CONCLUSION: CX3CR1+ CD4+ T cells characteristically increased in the peripheral blood and the affected tissues and were associated with an increase in the serum IgG4 levels of patients with IgG4-RD. This CD4 subset has a Th1/Tfh-like phenotype and a B cell helper function.

Development of a sandwich-type rat small intestine tissue sensor for detecting resveratrol and its receptors.

Resveratrol has a variety of biological functions, however, a limited number of studies have assessed its interaction with cell surface receptors. In this study, a sandwich-type rat small intestine tissue sensor (RSIT-sensor) was fabricated to detect the response current from receptor stimulation by different resveratrol concentrations via electrochemical workstation. The results showed that with detection limit of 1 × 10-13 mol/L, the maximum rate of change of the response current was found at the concentration of 8.5 × 10-12 mol/L, indicating that the resveratrol-related receptor was saturated. With comparing the response values of prepared biosensor and bare electrode with resveratrol, it can be concluded that the response value of small intestinal cells to resveratrol has obviously been amplified by the intracellular signal transmission system, and its magnification was about 100 times. In the current research, for the first time, kinetics of the interaction between resveratrol and its receptors and the transmission of signals to the body could be quantitatively measured by a biosensor. Our findings may provide new ideas for resveratrol-related receptor analysis, separation and purification, signal transmission, and evaluation of biological function.

Structural Conservation and Diversity of PilZ-Related Domains.

The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a variety of processes, including protein secretion, motility, cell development, and biofilm formation. c-di-GMP-dependent responses are often mediated by its binding to the cytoplasmic receptors that contain the PilZ domain. Here, we present comparative structural and sequence analysis of various PilZ-related domains and describe three principal types of them: (i) the canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a C-terminal alpha helix, (ii) an atypical PilZ domain that contains two extra alpha helices and forms stable tetramers, and (iii) divergent PilZ-related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as [D/N]hSXXG and show that the hydrophobic residue h of this motif interacts with a cluster of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe several novel PilZN-type domains that are fused to the canonical PilZ domains in specific taxa, such as spirochetes, actinobacteria, aquificae, cellulose-degrading clostridia, and deltaproteobacteria. We propose that the evolution of the three major groups of PilZ domains included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and other multidomain proteins; (ii) insertion of an ∼200-bp fragment, which resulted in the formation of tetramer-forming PilZ proteins; and (iii) tandem duplication of pilZ genes, which led to the formation of PilZ dimers and YcgR-like proteins.IMPORTANCE c-di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm formation, and virulence of many bacterial pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP through its RXXXR and [D/N]hSXXG motifs; some PilZ domains lack these motifs and are unable to bind c-di-GMP. We used structural and sequence analysis to assess the diversity of PilZ-related domains and define their common features. We show that the hydrophobic residue h in the second position of the second motif is highly conserved; it may serve as a readout for c-di-GMP binding. We describe three principal classes of PilZ-related domains, canonical, tetramer-forming, and divergent PilZ domains, and propose the evolutionary pathways that led to the emergence of these PilZ types.

Distinct Mycoplasma pneumoniae Interactions with Sulfated and Sialylated Receptors.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the conducting airways, causing bronchitis and atypical or "walking" pneumonia in humans. M. pneumoniae recognizes sialylated and sulfated oligosaccharide receptors to colonize the respiratory tract, but the contribution of the latter is particularly unclear. We used chamber slides coated with sulfatide (3-O-sulfogalactosylceramide) to provide a baseline for M. pneumoniae binding and gliding motility. As expected, M. pneumoniae bound to surfaces coated with sulfatide in a manner that was dependent on sulfatide concentration and incubation temperature and inhibited by competing dextran sulfate. However, mycoplasmas bound to sulfatide exhibited no gliding motility, regardless of receptor density. M. pneumoniae also bound lactose 3'-sulfate ligated to an inert polymer scaffold, and binding was inhibited by competing dextran sulfate. The major adhesin protein P1 mediates adherence to terminal sialic acids linked α-2,3, but P1-specific antibodies that blocked M. pneumoniae hemadsorption (HA) and binding to the sialylated glycoprotein laminin by 95% failed to inhibit mycoplasma binding to sulfatide, suggesting that P1 does not mediate binding to sulfated galactose. Consistent with this conclusion, the M. pneumoniae HA-negative mutant II-3 failed to bind to sialylated receptors but adhered to sulfatide in a temperature-dependent manner.

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

A Paradigm for Peptide Hormone-GPCR Analyses.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

The polymorphic proteins TgrB1 and TgrC1 function as a ligand-receptor pair in Dictyostelium allorecognition.

Allorecognition is a key factor in Dictyostelium development and sociality. It is mediated by two polymorphic transmembrane proteins, TgrB1 and TgrC1, which contain extracellular immunoglobulin domains. TgrB1 and TgrC1 are necessary and sufficient for allorecognition, and they carry out separate albeit overlapping functions in development, but their mechanism of action is unknown. Here, we show that TgrB1 acts as a receptor with TgrC1 as its ligand in cooperative aggregation and differentiation. The proteins bind each other in a sequence-specific manner; TgrB1 exhibits a cell-autonomous function and TgrC1 acts non-cell-autonomously. The TgrB1 cytoplasmic tail is essential for its function and it becomes phosphorylated upon association with TgrC1. Dominant mutations in TgrB1 activate the receptor function and confer partial ligand independence. These roles in development and sociality suggest that allorecognition is crucial in the integration of individual cells into a coherent organism.

Intrinsic and extrinsic mechanisms of dendritic morphogenesis.

The complex, branched morphology of dendrites is a cardinal feature of neurons and has been used as a criterion for cell type identification since the beginning of neurobiology. Regulated dendritic outgrowth and branching during development form the basis of receptive fields for neurons and are essential for the wiring of the nervous system. The cellular and molecular mechanisms of dendritic morphogenesis have been an intensely studied area. In this review, we summarize the major experimental systems that have contributed to our understandings of dendritic development as well as the intrinsic and extrinsic mechanisms that instruct the neurons to form cell type-specific dendritic arbors.

Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor.

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 µM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV.IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors.