Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes.

CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.

Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range.

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains.

Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.

Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.

Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.

Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses to the spike (S) protein monomer, S protein native trimeric form, or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection (n = 93) and in individuals enrolled in a postinfection seroprevalence population study (n = 578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein, or within a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute-infection-phase samples. Interestingly, compared to anti-S antibody responses, those against the N protein appear to wane in the postinfection cohort. Seroprevalence in a "positive patient contacts" group (n = 177) was underestimated by N protein assays by 10.9 to 32.2%, while the "randomly selected" general population group (n = 311) was reduced by up to 45% relative to the S protein assays. The overall reduction in seroprevalence targeting only anti-N antibodies for the total cohort ranged from 9.4 to 31%. Of note, the use of the S protein in its native trimer form was significantly more sensitive compared to monomeric S proteins. These results indicate that the assessment of anti-S IgG antibody responses against the native trimeric S protein should be implemented to estimate SARS-CoV-2 infections in population-based seroprevalence studies.IMPORTANCE In the present study, we have determined SARS-CoV-2-specific antibody responses in sera of acute and postinfection phase subjects. Our results indicate that antibody responses against viral S and N proteins were equally sensitive in the acute phase of infection, but that responses against N appear to wane in the postinfection phase where those against the S protein persist over time. The most sensitive serological assay in both acute and postinfection phases used the native S protein trimer as the binding antigen, which has significantly greater conformational epitopes for antibody binding compared to the S1 monomer protein used in other assays. We believe these results are extremely important in order to generate correct estimates of SARS-CoV-2 infections in the general population. Furthermore, the assessment of antibody responses against the trimeric S protein will be critical to evaluate the durability of the antibody response and for the characterization of a vaccine-induced antibody response.

Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC50 values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.

Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.

Pyridomycin bridges the NADH- and substrate-binding pockets of the enoyl reductase InhA.

Pyridomycin, a natural product with potent antituberculosis activity, inhibits a major drug target, the InhA enoyl reductase. Here, we unveil the co-crystal structure and unique ability of pyridomycin to block both the NADH cofactor- and lipid substrate-binding pockets of InhA. This is to our knowledge a first-of-a-kind binding mode that discloses a new means of InhA inhibition. Proof-of-principle studies show how structure-assisted drug design can improve the activity of new pyridomycin derivatives.

Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis.

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.

Discovery of benzothiazoles as antimycobacterial agents: Synthesis, structure-activity relationships and binding studies with Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose 2'-oxidase.

We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too.

The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron-sulfur cluster biogenesis and oxidative stress defence.

The complex multiprotein systems for the assembly of protein-bound iron-sulfur (Fe-S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe-S cluster biogenesis in other bacterial species. The ISC (iron-sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms. However, the cysteine desulfurase IscSMtb (Rv number Rv3025c; Mtb denotes M. tuberculosis) is conserved in this important pathogen. The present study demonstrates that deleting iscSMtb renders the cells microaerophilic and hypersensitive to oxidative stress. Moreover, the ∆iscSMtb mutant shows impaired Fe-S cluster-dependent enzyme activity, clearly indicating that IscSMtb is associated with Fe-S cluster assembly. An extensive interaction network of IscSMtb with Fe-S proteins was identified, suggesting a novel mechanism of sulfur transfer by direct interaction with apoproteins. Interestingly, the highly homologous IscS of Escherichia coli failed to complement the ∆iscSMtb mutant and showed a less diverse protein-interaction profile. To identify a structural basis for these observations we determined the crystal structure of IscSMtb, which mirrors adaptations made in response to an ISC operon devoid of IscU-like Fe-S cluster scaffold proteins. We conclude that in M. tuberculosis IscS has been redesigned during evolution to compensate for the deletion of large parts of the ISC operon.

Functional dissection of intersubunit interactions in the EspR virulence regulator of Mycobacterium tuberculosis.

The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.

Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA-independent virulence.

The type-VII ESX-1 secretion apparatus, encoded by the esx-1 genetic locus, is essential for the export of EsxA and EsxB, two major virulence factors of Mycobacterium tuberculosis. ESX-1 also requires the products of the unlinked espACD operon for optimal function and these proteins are considered integral parts of the secretion apparatus. Here we show that the espACD operon is not necessary for the secretion of EspB, another ESX-1 substrate, and this unimpeded secretion of EspB is associated with significant residual virulence. Upon further investigation, we found that purified EspB can facilitate M. tb virulence even in the absence of EsxA and EsxB, and may do so by binding the bioactive phospholipids phosphatidic acid and phosphatidylserine, both of which are potent bioactive molecules with prominent roles in eukaryotic cell signalling. Our findings provide new insights into the impact of the espACD operon on the ESX-1 apparatus and reveal a distinct virulence function for EspB with novel implications in M. tb-host interactions.

Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein.

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.

Peptide ligands stabilized by small molecules.

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50 mL and 600 mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20 mL and 400 mL, respectively.

Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research.

Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor-mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine IgG stripping from FBS and quality control Basic Protocol 2: AK23 hybridoma expansion and IgG production Basic Protocol 3: AK23 IgG purification Basic Protocol 4: AK23 IgG quality control Support Protocol 1: Detection of endotoxin levels Support Protocol 2: Detection and removal of mycoplasma.

Phenotypic profiling of Mycobacterium tuberculosis EspA point mutants reveals that blockage of ESAT-6 and CFP-10 secretion in vitro does not always correlate with attenuation of virulence.

The EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp(55) (W55) or Gly(57) (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe(50) (F50) and Lys(62) (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe(5) (F5) and Lys(41) (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.