Development of a rapid image-based high-content imaging screening assay to evaluate therapeutic antibodies against the monkeypox virus.

Antibody-based therapy is emerging as a critical therapeutic countermeasure to treat acute viral infections by offering rapid protection against clinical disease. The advancements in structural biology made it feasible to rationalize monoclonal antibodies (mAbs) by identifying key and, possibly, neutralizing epitopes of viral proteins for therapeutic purposes. A critical component in assessing mAbs during pandemics requires the development of rapid but detailed methods to detect and quantitate the neutralization activity. In this study, we developed and optimized two high-content image (HCI)-based assays: one to detect viral proteins by staining and the second to quantify cytopathic viral effects by a label-free phenotypic assay. These assays were employed to screen for therapeutic antibodies against the monkeypox virus (MPXV) using surrogate poxviruses such as vaccinia virus (VACV). Plaque-based neutralization results confirmed the HCI data. The phenotypic assay found pox virus-induced syncytia formation in various cells, and we were able to quantitate and use this phenotype to screen mAbs. The HCI identified several potent VACV-neutralizing antibodies that showed in vitro efficacy against both clades of MPXV. In addition, a combination study of ST-246/tecovirimat/TPOXX a single neutralizing antibody Ab-40, showed synergistic activity against VACV in an in-vitro neutralization assay. This rapid high-content method utilizing state-of-the-art technologies enabled the evaluation of hundreds of mAbs quickly to identify several potent anti-MPXV neutralizing mAbs for further development.

Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

In vitro microtubule severing assays.

Microtubules are rigid and highly dynamic cellular polymers essential for intracellular transport, cell division and differentiation. Their stability is tightly regulated by a vast array of cellular factors. In vitro microtubule assays have proven to be powerful tools for deciphering the mechanism of microtubule dynamics regulators such as molecular motors and microtubule associated proteins. In this chapter we focus on microtubule severing enzymes that use the energy of ATP hydrolysis to introduce internal breaks in the microtubule lattice. We present a detailed protocol for a light microscopy based in vitro microtubule severing assay that was instrumental in the identification and characterization of these enzymes.

New active HIV-1 protease inhibitors derived from 3-hexanol: conformation study of the free inhibitors in crystalline state and in complex with the enzyme.

Four novel linear non-peptidic HIV-1 protease inhibitors derived from 2,5-diamino-1,6-diphenyl-3-hexanol were synthesized and characterized. All of them exhibit tight binding to HIV-1 protease, with inhibition constants K(i) in the range 20 pm-5 nm. The investigated inhibitors were crystallized, and their crystal structures were determined by X-ray diffraction. In all cases, the conformations found in the crystalline state differ significantly from the conformations obtained by computational docking of the inhibitor in the binding cleft of native HIV-1 protease. Owing to the prevalence of hydrophobic substituents in all these inhibitors, the conformational mobility in water solution is restricted to their compact forms. The spectrum of low-energy conformations in solution dramatically changes during the formation of inhibitor crystals (phenyl ring stacking as a leading motif) or during the formation of a complex with HIV-1 protease (elongated conformation suitable to fit the enzyme pockets as a factor responsible for tight binding). High conformational flexibility and low conformational stress in the molecules of these inhibitors most likely increase their biological activity in comparison with more rigid compounds.

Organized living: formation mechanisms and functions of plasma membrane domains in yeast.

Plasma membrane proteins and lipids organize into lateral domains of specific composition. Domain formation is achieved by a combination of lipid-lipid and lipid-protein interactions, membrane-binding protein scaffolds and protein fences. The resulting domains function in membrane protein turnover and homeostasis, as well as in cell signaling. We review the mechanisms generating plasma membrane domains and the functional consequences of this organization, focusing on recent findings from research on the yeast model system.

Eisosome proteins assemble into a membrane scaffold.

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.

Eisosome-driven plasma membrane organization is mediated by BAR domains.

Plasma membranes are organized into domains of different protein and lipid composition. Eisosomes are key complexes for yeast plasma membrane organization, containing primarily Pil1 and Lsp1. Here we show that both proteins consist mostly of a banana-shaped BAR domain common to membrane sculpting proteins, most similar to the ones of amphiphysin, arfaptin 2 and endophilin 2. Our data reveal a previously unrecognized family of BAR-domain proteins involved in plasma membrane organization.

Structural investigation of biologically active phenolic compounds isolated from European tree species.

X-ray structures of two compounds isolated from wood knots of coniferous trees, namely dihydrokaempferol (3,5,8,13-tetrahydroxyflavanon) and lariciresinol (3,14-dimetoxy-7,10-epoxylignan-4,15,19-triol), are presented here. Diffraction data for the Dihydrokaempferol crystals were collected on a CAD4 diffractometer and on a synchrotron for the lariciresinol crystal. The investigated compounds inhibit lipid peroxidation and lariciresinol is additionally a good scavenger of superoxide radicals. The structural data presented in this work provide a useful basis for designing more active compounds with potential use as antioxidants.

Atomic-resolution crystal structure of the antiviral lectin scytovirin.

The crystal structures of the natural and recombinant antiviral lectin scytovirin (SVN) were solved by single-wavelength anomalous scattering and refined with data extending to 1.3 A and 1.0 A resolution, respectively. A molecule of SVN consists of a single chain 95 amino acids long, with an almost perfect sequence repeat that creates two very similar domains (RMS deviation 0.25 A for 40 pairs of Calpha atoms). The crystal structure differs significantly from a previously published NMR structure of the same protein, with the RMS deviations calculated separately for the N- and C-terminal domains of 5.3 A and 3.7 A, respectively, and a very different relationship between the two domains. In addition, the disulfide bonding pattern of the crystal structures differs from that described in the previously published mass spectrometry and NMR studies.

Crystallographic studies of the complexes of antiviral protein griffithsin with glucose and N-acetylglucosamine.

Crystal structures of complexes of an antiviral lectin griffithsin (GRFT) with glucose and N-acetylglucosamine were solved and refined at high resolution. In both complexes, all six monosaccharide-binding sites of GRFT were occupied and the mode of binding was similar to that of mannose. In our previous attempts to obtain a complex with N-acetylglucosamine by soaking, only a single site was occupied; thus, cocrystallization was clearly superior despite lower concentration of the ligand. Isothermal titration calorimetric experiments with N-acetylglucosamine, glucose, and mannose provided enthalpic evidence of distinct binding differences between the three monosaccharides. A comparison of the mode of binding of different monosaccharides is discussed in the context of the antiviral activity of GRFT, based on specific binding to high-mannose-containing complex carbohydrates found on viral envelopes.

Crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin.

The mode of binding of oligosaccharides to griffithsin, an antiviral lectin from the red alga Griffithsia sp., was investigated by a combination of X-ray crystallography, isothermal titration calorimetry, and molecular modeling. The structures of complexes of griffithsin with 1-->6alpha-mannobiose and with maltose were solved and refined at the resolution of 2.0 and 1.5 A, respectively. The thermodynamic parameters of binding of 1-->6alpha-mannobiose, maltose, and mannose to griffithsin were determined. Binding profiles of 1-->6alpha-mannobiose and mannose were similar with Kd values of 83.3 microM and 102 microM, respectively. The binding of maltose to griffithsin was significantly weaker, with a fourfold lower affinity (Kd = 394 microM). In all cases the binding at 30 degrees C was entropically rather than enthalpically driven. On the basis of the experimental crystal structures, as well as on previously determined structures of complexes with monosaccharides, it was possible to create a model of a tridentate complex of griffithsin with Man9GlcNAc2, a high mannose oligosaccharide commonly found on the surface of viral glycoproteins. All shorter oligomannoses could be modeled only as bidentate or monodentate complexes with griffithsin. The ability to mediate tight multivalent and multisite interactions with high-mannose oligosaccharides helps to explain the potent antiviral activity of griffithsin.

Structural studies of algal lectins with anti-HIV activity.

A number of antiviral lectins, small proteins that bind carbohydrates found on viral envelopes, are currently in pre-clinical trials as potential drugs for prevention of transmission of human immunodeficiency virus (HIV) and other enveloped viruses, such as the Ebola virus and the coronavirus responsible for severe acute respiratory syndrome (SARS). Lectins of algal origin whose antiviral properties make them candidate agents for prevention of viral transmission through topical applications include cyanovirin-N, Microcystis viridis lectin, scytovirin, and griffithsin. Although all these proteins exhibit significant antiviral activity, their structures are unrelated and their mode of binding of carbohydrates differs significantly. This review summarizes the current state of knowledge of the structures of algal lectins, their mode of binding of carbohydrates, and their potential medical applications.

Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding.

The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 A resolution for the free protein and 0.94 A for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two beta strands of one molecule complete a beta prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.