Full-length Structure Research Articles

Elucidation of the DNA-Binding Activity of VirF from Shigella flexneri for the icsA and rnaG Promoters and Characterization of the N-Terminal Domain To Identify Residues Crucial for Dimerization.

A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid β-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

1H, 13C, 15N Backbone and sidechain chemical shift assignments of the C-terminal domain of human UDP-glucuronosyltransferase 2B17 (UGT2B17-C).

UDP-glucuronosyltransferases are the principal enzymes involved in the glucuronidation of metabolites and xenobiotics for physiological clearance in humans. Though glucuronidation is an indispensable process in the phase II metabolic pathway, UGT-mediated glucuronidation of most prescribed drugs (> 55%) and clinical evidence of UGT-associated drug resistance are major concerns for therapeutic development. While UGTs are highly conserved enzymes, they manifest unique substrate and inhibitor specificity which is poorly understood given the dearth of experimentally determined full-length structures. Such information is important not only to conceptualize their specificity but is central to the design of inhibitors specific to a given UGT in order to avoid toxicity associated with pan-UGT inhibitors. Here, we provide the 1H, 13C and 15N backbone (~ 90%) and sidechain (~ 62%) assignments for the C-terminal domain of UGT2B17, which can be used to determine the molecular binding sites of inhibitor and substrate, and to understand the atomic basis for inhibitor selectivity between UGT2B17 and other members of the UGT2B subfamily. Given the physiological relevance of UGT2B17 in the elimination of hormone-based cancer drugs, these assignments will contribute towards dissecting the structural basis for substrate specificity, selective inhibitor recognition and other aspects of enzyme activity with the goal of selectively overcoming glucuronidation-based drug resistance.

Mapping the binding site of NS309, a superagonistic positive gating modulator for the calcium-activated potassium channel KCa3.1.

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is expressed in erythrocytes, vascular endothelial cells, and immune cells. KCa3.1 is involved in a wide variety of disease pathologies, suggesting that both positive and negative gating modulators would be of interest as pharmacological tools and potential drugs. NS309 is the most potent positive gating modulator for KCa3.1, with an EC50 of 75 nM, and interestingly, it is capable of increasing channel activity even at saturating calcium concentrations. However, its binding site is currently unknown. Previous X-ray crystallographic and solution-state NMR studies suggested that NS309 was binding between calmodulin's N-lobe and the C-terminal region of the calmodulin binding domain and interacting with residues M51 and R372. Unfortunately, the crystal dimer used in these studies was later found to be an artifact and disproved when the full-length crystal structure of KCa3.1 was published in 2018. This full-length structure suggested that the “real” binding site for positive gating modulators was located within the interface between calmodulin's N-lobe and the S4-S5 linker, a hypothesis we have now confirmed via site-directed mutagenesis and whole-cell patch clamp. In addition, we have also been investigating a second binding pocket between the distal end of the S4-S5 intracellular loop and the S6 transmembrane segment (a site highlighted by Schroedinger's SiteMap software). And while NS309 does not seem to be binding in this second pocket, several mutations within this pocket cause the channel to become hypersensitive to calcium, indicating that this pocket could serve as another functional binding site for positive gating channel modulators.

Computational modeling and molecular mapping of serine protease inhibitor family A5 (SERPINA5) structure, associated with tau expression and Alzheimer's disease.

Serine Protease Inhibitor Family AS (SERPINA5), also known as PCI (Protein C Inhibitor) or PAl3 (plasminogen activator inhibitor 3), is a member of the serine proteinase inhibitor family. Our previous studies have shown that increased expression of the SERPINA5 gene is associated with tau expression in the brain and Alzheimer's disease. We hypothesize selective targeting of SERPINA5 has potential as a therapy for Alzheimer's disease. Since the full-length 3D human SERPINA5 structure is still unknown, we performed computational modeling in silico using Schrodinger Suite 2022-2 (Prime software), AlphaFold 2 and OpenFold (trainable PyTorch reproduction of DeepMind's AlphaFold). Molecular mapping was used to identify binding hot spots, which are regions of the protein surface that can bind small molecules or drug fragments and was calculated by ATLAS (FTMap) software. The best structures with different conformational states were selected and molecular mapping was performed for each of them. The druggability hot spots were determined, then ranked according to their ability to bind small molecules and peptides on the SERPINA5 protein surface. These results will be used in molecular docking, molecular dynamics simulations and biological testing research.

Structural and biophysical studies of the T-box riboswitch.

Riboswitches are noncoding structured RNAs that bind molecules and regulate gene expression in bacteria. They are commonly found in the 5' untranslated regions of bacterial messenger RNAs. T-box riboswitches bind to specific transfer RNAs (tRNAs), sense the aminoacylation state of those tRNAs, and switch conformations to regulate amino-acid metabolism and nutritional homeostasis. T-boxes are redundantly employed in bacteria to control many important operons inside a single cell, making them attractive targets for antibiotic development. Structural and biophysical characterization of this biomolecule would aid the search for T-box riboswitch inhibitors. Although high-resolution structures of some T-boxes from the glycine and atypical classes have been solved, the structure of a full-length typical T-box, the most abundant in nature, has not been achieved to date. This project has made advancements to obtain the first full length structure of a typical T-box riboswitch in complex with its cognate tRNA substrate by single particle cryo-electron microscopy. Bioinformatics analyses were performed to identify putative T-box sequences with high structural stability. This information was used to construct medium-throughput plasmid libraries of T-box-tRNA pairs for in vitro transcription. T-box-tRNA complexes were then isolated from these transcription reactions. To identify the particular sequences that formed stable complexes under the working conditions, an EMSA based assay using fluorescently labelled DNA probes was developed. The integrity of T-box-tRNA complexes was validated by dynamic light scattering and negative stain electron microscopy. These candidate sequences are currently being probed for cryo-electron microscopy studies. Moreover, this project also aims to characterize the binding kinetics of the translational class of T-boxes to tRNA using single-molecule FRET (smFRET). How the biophysical effects of this interaction have been adapted by Actinobacteria to regulate gene expression at the translational level has been elucidated by combining smFRET studies with strategic mutagenesis.

Structure modelling of odorant receptor from Aedes aegypti and identification of potential repellent molecules

Odorant receptors (ORs) are important class of proteins involved in olfactory behaviour of insects. These are GPCR-like heptahelical transmembrane proteins with inverted topology compared to GPCR and require a co-receptor (ORco) for their function. OR function can be modulated through small molecules and negative modulation can be beneficial in case of disease vectors like Aedes aegypti. OR4 of A. aegypti is implicated in host recognition through human odour. Aedes aegypti is a vector for viruses that spread diseases like dengue, Zika and Chikungunya. In this study, we have attempted to model the full-length structure of OR4 and the ORco of A. aegypti due to lack of experimental structure. Further, we have screened a library of natural compounds (>0.3 million) along with known repellent molecules against ORco and OR4. Many natural compounds, including those from plants like Ocimum tenuiflorum (Holy Basil) and Piper nigrum (Black pepper), were found to have better binding affinity towards ORco compared to known repellents like DEET providing an alternative to existing repellent molecules. For specific inhibitor of OR4, several natural compounds (including those from plant like Mulberry) were identified. Further, we have utilized multiple docking approaches and conservation analysis to understand the interaction between OR4 and ORco. It was observed that the residues from the seventh transmembrane helix of OR4 and pore forming helix of ORco could play an important role along with known intracellular loop 3 residues in mediating the heteromer formation of OR and ORco.

Druggable Pockets at the RNA Interface Region of Influenza A Virus NS1 Protein Are Conserved across Sequence Variants from Distinct Subtypes.

Influenza A viruses still represent a major health issue, for both humans and animals. One of the main viral proteins of interest to target is the NS1 protein, which counters the host immune response and promotes viral replication. NS1 is a homodimer composed of a dimeric RNA-binding domain (RBD), which is structurally stable and conserved in sequence, and two effector domains that are tethered to the RBD by linker regions. This linker flexibility leads to NS1 polymorphism and can therefore exhibit different forms. Previously, we identified a putative drug-binding site, located in the RBD interface in a crystal structure of NS1. This pocket could be targeted to block RNA binding and inhibit NS1 activities. The objective of the present study is to confirm the presence of this druggable site, whatever the sequence variants, in order to develop a universal therapeutic compound that is insensitive to sequence variations and structural flexibility. Using a set of four NS1 full-length structures, we combined different bioinformatics approaches such as pocket tracking along molecular dynamics simulations, druggability prediction and classification. This protocol successfully confirmed a frequent large binding-site that is highly druggable and shared by different NS1 forms, which is promising for developing a robust NS1-targeted therapy.

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction.

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

In silico modeling human VPS13 proteins associated with donor and target membranes suggests lipid transfer mechanisms.

The VPS13 protein family constitutes a novel class of bridge-like lipid transferases. Autosomal recessive inheritance of mutations in VPS13 genes is associated with the development of neurodegenerative diseases in humans. Bioinformatic approaches previously recognized the domain architecture of these proteins. In this study, we model the first ever full-length structures of the four human homologs VPS13A, VPS13B, VPS13C, and VPS13D in association with model membranes, to investigate their lipid transfer ability and potential structural association with membrane leaflets. We analyze the evolutionary conservation and physicochemical properties of these proteins, focusing on conserved C-terminal amphipathic helices that disturb organelle surfaces and that, adjoined, resemble a traditional Venetian gondola. The gondola domains share significant structural homology with lipid droplet surface-binding proteins. We introduce in silico protein-membrane models displaying the mode of association of VPS13A, VPS13B, VPS13C, and VPS13D to donor and target membranes, and present potential models of action for protein-mediated lipid transfer.

A new polymorphism of human amylin fibrils with similar protofilaments and a conserved core.

Pancreatic amyloid deposits composed of a fibrillar form of the human islet amyloid polypeptide (hIAPP) are the pathological hallmark of type 2 diabetes (T2D). Although various cryo-EM structures of polymorphic hIAPP fibrils were reported, the underlying polymorphic mechanism of hIAPP remains elusive. Meanwhile, the structure of hIAPP fibrils with all residues visible in the fibril core is not available. Here, we report the full-length structures of two different polymorphs of hIAPP fibrils, namely slim form (SF, dimer) and thick form (TF, tetramer), formed in a salt-free environment, which share a similar ζ-shaped protofilament but differ in inter-protofilament interfaces. In the absence of salt, electrostatic interactions were found to play a dominant role in stabilizing the fibril structure, suggesting an antagonistic effect between electrostatic and hydrophobic interactions in different salt concentrations environments. Our results shed light on understanding the mechanism of amyloid fibril polymorphism.

Cryo-EM structure of the diapause chaperone artemin.

The protein artemin acts as both an RNA and protein chaperone and constitutes over 10% of all protein in Artemia cysts during diapause. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04Å resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage but in a different configuration than previously hypothesized with molecular modeling. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

Cryo-EM structure of the human sodium-chloride cotransporter NCC.

The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. Here, we present the full-length structure of human NCC, with 2.9 Å for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain. NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in our structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules are indicated to participate in the coordination of ions and TM coupling. Together with transport activity assays, our structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on NCC.

Refinement of SARS-CoV-2 envelope protein structure in a native-like environment by molecular dynamics simulations.

COVID-19 has become an unprecedented threat to human health. The SARS-CoV-2 envelope (E) protein plays a critical role in the viral maturation process and pathogenesis. Despite intensive investigation, its structure in physiological conditions remains mysterious: no high-resolution full-length structure is available and only an NMR structure of the transmembrane (TM) region has been determined. Here, we present a refined E protein structure, using molecular dynamics (MD) simulations to investigate its structure and dynamics in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer system. Our initial homology model based upon the SARS-CoV E protein structure is shown to be unstable in the lipid bilayer, and the H3 helices tend to move away from the membrane center to the membrane-water interface. A more stable model was developed by replacing all H3 helices with the fully equilibrated H3 structure sampled in the MD simulations. This refined model exhibited more favorable contacts with lipids and water than the original homology model and induced local membrane curvature, decreasing local lipid order. Interestingly, the pore radius profiles showed that the channel in both homology and refined models remained in a closed state throughout the simulations. We also demonstrated the utility of this structure to develop anti-SARS-CoV-2 drugs by docking a library of FDA-approved, investigational, and experimental drugs to the refined E protein structure, identifying 20 potential channel blockers. This highlights the power of MD simulations to refine low-resolution structures of membrane proteins in a native-like membrane environment, shedding light on the structural features of the E protein and providing a platform for the development of novel antiviral treatments.

VP.65 Screening of small molecules for activation of GNE protein carrying non-catalytic site mutation based on molecular docking simulation

GNE myopathy is an autosomal recessive distal myopathy caused by pathogenic variants in <i>GNE</i> gene, which encodes a protein having two enzymatic activities, UDP-GlcNAc 2-epimerase and ManNAc kinase on sialic acid biosynthesis. We have identified a variant, c.620A>T (p.D207V), with which majority of the homozygotes are healthy, while compound heterozygotes develop myopathy. Notwithstanding, the identified three affected homozygotes had no additional variation in the <i>GNE</i> gene nor in genes in sialic acid biosynthetic pathway, while their sialylation was mildly, but certainly decreased. We hypothesized this discrepancy can be explainable by partial failure of GNE oligomerization. The constructed full-length GNE structure by Alphafold2 indicated that Asp207 locates midway between each domain and near subunit interface, but not in catalytic site. The mutant was predicted to cause no severe changes in a whole structure, though the change of polarity of the mutated residue and failure of oligomerization are hypothesized to affect intramolecular and inter-subunit ManNAc transfer between domains. Since this pathogenic variant is in non-catalytic site and did not cause drastic structural changes, ManNAc production has possibility to be pharmacologically recovered by small molecules. Here, we performed <i>in silico</i> prediction-based screening to identify the drugs that activate GNE. First, we targeted a deep narrow cavity found in the back of the allosteric site, to find small molecules that interfere with negative feedback inhibition. We then performed docking simulation of 8042 known compounds in Drugbank database to the cavity, and selected well-fitted 8 compounds. We measured the effect of the compounds on ManNAc production. However, no compounds could activate it nor repress inhibitory effect of CMP-NeuAc. In this presentation, we will discuss brand-new approach to find activators for endogenous mutated GNE protein using structural information.

In-solution structure and oligomerization of human histone deacetylase 6 - an integrative approach.

Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full-length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in-solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomerization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure of full-length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.

AlphaFold and Structural Mass Spectrometry Enable Interrogations on the Intrinsically Disordered Regions in Cyanobacterial Light-harvesting Complex Phycobilisome

Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.

Homology modeling of Forkhead box protein C2: identification of potential inhibitors using ligand and structure-based virtual screening.

Overexpression of Forkhead box protein C2 (FOXC2) has been associated with different types of carcinomas. FOXC2 plays an important role in the initiation and maintenance of the epithelial-mesenchymal transition (EMT) process, which is essential for the development of higher-grade tumors with an enhanced ability for metastasis. Thus, FOXC2 has become a therapeutic target for the development of anticancer drugs. MC-1-F2, the only identified experimental inhibitor of FOXC2, interacts with the full length of FOXC2. However, only the DNA-binding domain (DBD) of FOXC2 has resolved crystal structure. In this work, a three-dimensional (3D) structure of the full-length FOXC2 using homology modeling was developed and used for structure-based drug design (SBDD). The quality of this 3D model of the full-length FOXC2 was evaluated using MolProbity, ERRAT, and ProSA modules. Molecular dynamics (MD) simulation was also carried out to verify its stability. Ligand-based drug design (LBDD) was carried out to identify similar analogues for MC-1-F2 against 15 million compounds from ChEMBL and ZINC databases. 792 molecules were retrieved from this similarity search. De novo SBDD was performed against the full-length 3D structure of FOXC2 through homology modeling to identify novel inhibitors. The combination of LBDD and SBDD helped in gaining a better insight into the binding of MC-1-F2 and its analogues against the full length of the FOXC2. The binding free energy of the top hits was further investigated using MD simulations and MM/GBSA calculations to result in eight promising hits as lead compounds targeting FOXC2.

Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+ -ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the β-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the β-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+ -ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.

Using the multi-omics approach to reveal the silk composition in Plectrocnemia conspersa.

Similar to Lepidoptera, the larvae of Trichoptera are also capable of producing silk. Plectrocnemia conspersa, a predatory species belonging to the suborder Annulipalpia, builds massive silken retreats with preycapturing nets. In this study, we describe the silk glands of P. conspersa and use the multi-omics methods to obtain a complete picture of the fiber composition. A combination of silk gland-specific transcriptome and proteomic analyses of the spun-out fibers yielded 27 significant candidates whose full-length sequences and gene structures were retrieved from the publicly available genome database. About one-third of the candidates were completely novel proteins for which there are no described homologs, including a group of five pseudofibroins, proteins with a composition similar to fibroin heavy chain. The rest were homologs of lepidopteran silk proteins, although some had a larger number of paralogs. On the other hand, P. conspersa fibers lacked some proteins that are regular components in moth silk. In summary, the multi-omics approach provides an opportunity to compare the overall composition of silk with other insect species. A sufficient number of such studies will make it possible to distinguish between the basic components of all silks and the proteins that represent the adaptation of the fibers for specific purposes or environments.