Expanding the insect defensin landscape.

In this issue of Structure, Walker et al.1 determined the NMR structure of a recently discovered defensin, Pp19, from the venom of an assassin bug. This peptide adopts an α-defensin-like structure, which had not been observed in insects before. Unlike mammalian α-defensins, which are generally antimicrobial, Pp19 has insecticidal activity.

Ping, pong, and freeze: Structural insights into the inhibition of ceramide synthase by Fumonisin B1.

Fumonisin B1 (FB1) targets sphingolipid biosynthesis, inhibiting ceramide synthases. In this issue of Structure, Zhang et al.1 determined the cryoelectron microscopic structures of yeast ceramide synthase in complex with FB1 and its acylated derivative, acyl-FB1, revealing a two-step "ping-pong" mechanism for the N-acylation of FB1 and how it inhibits ceramide synthase.

Structural review of SARS-CoV-2 antiviral targets.

The coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents the most disastrous infectious disease pandemic of the past century. As a member of the Betacoronavirus genus, the SARS-CoV-2 genome encodes a total of 29 proteins. The spike protein, RNA-dependent RNA polymerase, and proteases play crucial roles in the virus replication process and are promising targets for drug development. In recent years, structural studies of these viral proteins and of their complexes with antibodies and inhibitors have provided valuable insights into their functions and laid a solid foundation for drug development. In this review, we summarize the structural features of these proteins and discuss recent progress in research regarding therapeutic development, highlighting mechanistically representative molecules and those that have already been approved or are under clinical investigation.

Molecular Modeling Methods in the Development of Affine and Specific Protein-Binding Agents.

High-affinity and specific agents are widely applied in various areas, including diagnostics, scientific research, and disease therapy (as drugs and drug delivery systems). It takes significant time to develop them. For this reason, development of high-affinity agents extensively utilizes computer methods at various stages for the analysis and modeling of these molecules. The review describes the main affinity and specific agents, such as monoclonal antibodies and their fragments, antibody mimetics, aptamers, and molecularly imprinted polymers. The methods of their obtaining as well as their main advantages and disadvantages are briefly described, with special attention focused on the molecular modeling methods used for their analysis and development.

@TOME 3.0: Interfacing Protein Structure Modeling and Ligand Docking.

Knowledge of protein-ligand complexes is essential for efficient drug design. Virtual docking can bring important information on putative complexes but it is still far from being simultaneously fast and accurate. Receptors are flexible and adapt to the incoming small molecules while docking is highly sensitive to small conformational deviations. Conformation ensemble is providing a mean to simulate protein flexibility. However, modeling multiple protein structures for many targets is seldom connected to ligand screening in an efficient and straightforward manner. @TOME-3 is an updated version of our former pipeline @TOME-2, in which protein structure modeling is now directly interfaced with flexible ligand docking. Sequence-sequence profile comparisons identify suitable PDB templates for structure modeling and ligands from these templates are used to deduce binding sites to be screened. In addition, bound ligand can be used as pharmacophoric restraint during the virtual docking. The latter is performed by PLANTS while the docking poses are analysed through multiple chemoinformatics functions. This unique combination of tools allows rapid and efficient ligand docking on multiple receptor conformations in parallel. @TOME-3 is freely available on the web at https://atome.cbs.cnrs.fr.

GalaxySagittarius-AF: Predicting Targets for Drug-Like Compounds in the Extended Human 3D Proteome.

In recent years, advancements in deep learning techniques have significantly expanded the structural coverage of the human proteome. GalaxySagittarius-AF translates these achievements in structure prediction into target prediction for druglike compounds by incorporating predicted structures. This web server searches the database of human protein structures using both similarity- and structure-based approaches, suggesting potential targets for a given druglike compound. In comparison to its predecessor, GalaxySagittarius, GalaxySagittarius-AF utilizes an enlarged structure database, incorporating curated AlphaFold model structures alongside their binding sites and ligands, predicted using an updated version of GalaxySite. GalaxySagittarius-AF covers a large human protein space compared to many other available computational target screening methods. The structure-based prediction method enhances the use of expanded structural information, differentiating it from other target prediction servers that rely on ligand-based methods. Additionally, the web server has undergone enhancements, operating two to three times faster than its predecessor. The updated report page provides comprehensive information on the sequence and structure of the predicted protein targets. GalaxySagittarius-AF is accessible at https://galaxy.seoklab.org/sagittarius_af without the need for registration.

3dRNA/DNA: 3D Structure Prediction from RNA to DNA.

There is an increasing need for determining 3D structures of DNAs, e.g., for increasing the efficiency of DNA aptamer selection. Recently, we have proposed a computational method of 3D structure prediction of DNAs, called 3dDNA, which has been integrated into our original web server 3dRNA, now renamed 3dRNA/DNA (http://biophy.hust.edu.cn/new/3dRNA). Currently, 3dDNA can only output the predicted DNA 3D structures for users but cannot rank them as an energy function for assessing DNA 3D structures is still lacking. Here, we first provide a brief introduction to 3dDNA and then introduce a new energy function, 3dDNAscore, for the assessment of DNA 3D structures. 3dDNAscore is an all-atom knowledge-based potential by integrating 86 atomic types from nucleic acids. Benchmarks demonstrate that 3dDNAscore can effectively identify near-native structures from the decoys generated by 3dDNA, thus enhancing the completeness of 3dDNA.

E-pRSA: Embeddings Improve the Prediction of Residue Relative Solvent Accessibility in Protein Sequence.

Knowledge of the solvent accessibility of residues in a protein is essential for different applications, including the identification of interacting surfaces in protein-protein interactions and the characterization of variations. We describe E-pRSA, a novel web server to estimate Relative Solvent Accessibility values (RSAs) of residues directly from a protein sequence. The method exploits two complementary Protein Language Models to provide fast and accurate predictions. When benchmarked on different blind test sets, E-pRSA scores at the state-of-the-art, and outperforms a previous method we developed, DeepREx, which was based on sequence profiles after Multiple Sequence Alignments. The E-pRSA web server is freely available at https://e-prsa.biocomp.unibo.it/main/ where users can submit single-sequence and batch jobs.

Pretrainable geometric graph neural network for antibody affinity maturation.

Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50 values of the designed antibody mutants are decreased by up to 17 fold, and KD values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.

​Fusarium Protein Toolkit: a web-based resource for structural and variant analysis of Fusarium species.

BACKGROUND: ​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies. DESCRIPTION: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. CONCLUSION: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.

Exploring the structural landscape of DNA maintenance proteins.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

Unveiling the solution structure of a DNA duplex with continuous silver-modified Watson-Crick base pairs.

The challenge of transforming organized DNA structures into their metallized counterparts persists in the scientific field. In this context, utilizing DNA molecules modified with 7-deazapurine, provides a transformative solution. In this study, we present the solution structure of a DNA duplex that can be transformed into its metallized equivalent while retaining the natural base pairing arrangement through the creation of silver-modified Watson-Crick base pairs. Unlike previously documented X-ray structures, our research demonstrates the feasibility of preserving the intrinsic DNA self-assembly while incorporating AgI into the double helix, illustrating that the binding of silver does not disrupt the canonical base-pairing organization. Moreover, in our case, the uninterrupted AgI chain deviates from forming conventional straight linear chains; instead, it adheres to a helical arrangement dictated by the underlying DNA structure. This research challenges conventional assumptions and opens the door to precisely design structures based on the organization of highly stable Ag-DNA assemblies.

A bitopic agonist bound to the dopamine 3 receptor reveals a selectivity site.

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity and improve current medications by reducing off-target side effects. However, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R:GαOßγ complex bound to the D3R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.

Cryo-EM structures of a mycobacterial ABC transporter that mediates rifampicin resistance.

Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, Mycobacterium tuberculosis (Mtb), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Mtb Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance.

Bacterial ß-carbonic anhydrases.

ß-Carbonic anhydrases (ß-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. ß-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. ß-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the ß-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II ß-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.

Association mechanism of bicalutamide and human serum albumin for potential clinical implications.

The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104 J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.

Exploring protein structural ensembles: Integration of sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling methods.

Under physiological conditions, proteins continuously undergo structural fluctuations on different timescales. Some conformations are only sparsely populated, but still play a key role in protein function. Thus, meaningful structure-function frameworks must include structural ensembles rather than only the most populated protein conformations. To detail protein plasticity, modern structural biology combines complementary experimental and computational approaches. In this review, we survey available computational approaches that integrate sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling techniques to derive all-atom structural models of rare protein conformations. We also propose strategies to increase the reliability and improve efficiency using deep learning approaches, thus advancing the field of integrative structural biology.

Neurotransmitter recognition by human vesicular monoamine transporter 2.

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design.

Structural roles of Ump1 and ß-subunit propeptides in proteasome biogenesis.

The yeast pre1-1(ß4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with ß2- and ß5-propeptides, which together fill most of the antechambers between the α- and ß-rings. The ß5-propeptide is unprocessed and separates Ump1 from ß6 and ß7. The ß2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and ß3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, ß2 is activated first enabling processing of ß1-, ß6-, and ß7-propeptides. Subsequent maturation of ß5 and ß1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.

Non-standard amino acid incorporation into thiol dioxygenases.

Thiol dioxygenases (TDOs) are non­heme Fe(II)­dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common ß­barrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3­His iron­binding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Site­directed mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of non­standard amino acid incorporation.