Protein-ligand interactions represent an essential step to understand the bases of molecular recognition, an intense field of research in many scientific areas. Structural biology has played a central role in unveiling protein-ligand interactions, but current techniques are still not able to reliably describe the interactions of ligands with highly flexible regions. In this work, we explored the capacity of AlphaFold2 (AF2) to estimate the presence of interactions between ligands and residues belonging to disordered regions. As these interactions are missing in the crystallographic-derived structures, we called them "ghost interactions". Using a set of protein structures experimentally obtained after AF2 was trained, we found that the obtained models are good predictors of regions associated with order-disorder transitions. Additionally, we found that AF2 predicts residues making ghost interactions with ligands, which are mostly buried and show differential evolutionary conservation with the rest of the residues located in the flexible region. Our findings could fuel current areas of research that consider, given their biological relevance and their involvement in diseases, intrinsically disordered proteins as potentially valuable targets for drug development.

Precise regulation of protein kinase activity is crucial in cell functions, and its loss is implicated in various diseases. The kinase activity is regulated by interconverting active and inactive states in the conformational landscape. However, how protein kinases switch conformations in response to different signals such as the binding at distinct sites remains incompletely understood. Here, we predict the binding mode for the peptide substrate to Src tyrosine kinase using enhanced conformational sampling simulations (totaling 24 μs) and then investigate changes in the conformational landscape upon substrate binding by conducting unbiased molecular dynamics simulations (totaling 50 μs) initiated from the apo and substrate-bound forms. Unexpectedly, the peptide substrate binding significantly facilitates the transitions from active to inactive conformations in which the αC helix is directed outward, the regulatory spine is broken, and the ATP-binding domain is perturbed. We also explore an underlying residue-contact network responsible for the allosteric conformational changes. Our results are in accord with the recent experiments reporting the negative cooperativity between the peptide substrate and ATP binding to tyrosine kinases and will contribute to advancing our understanding of the regulation mechanisms for kinase activity.

Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously "undruggable" protein targets, paving the way for effective interventions in disease.

N6-methyladenosine (m6A) modification, a prevalent epigenetic mark in eukaryotic cells, is crucial in regulating gene expression and RNA metabolism. Accurately identifying m6A modification sites is essential for understanding their functions within biological processes and the intricate mechanisms that regulate them. Recent advances in high-throughput sequencing technologies have enabled the generation of extensive datasets characterizing m6A modification sites at single-nucleotide resolution, leading to the development of computational methods for identifying m6A RNA modification sites. However, most current methods focus on specific cell lines, limiting their generalizability and practical application across diverse biological contexts. To address the limitation, we propose MST-m6A, a novel approach for identifying m6A modification sites with higher accuracy across various cell lines and tissues. MST-m6A utilizes a multi-scale transformer-based architecture, employing dual k-mer tokenization to capture rich feature representations and global contextual information from RNA sequences at multiple levels of granularity. These representations are then effectively combined using a channel fusion mechanism and further processed by a convolutional neural network to enhance prediction accuracy. Rigorous validation demonstrates that MST-m6A significantly outperforms conventional machine learning models, deep learning models, and state-of-the-art predictors. We anticipate that the high precision and cross-cell-type adaptability of MST-m6A will provide valuable insights into m6A biology and facilitate advancements in related fields. The proposed approach is available at https://github.com/cbbl-skku-org/MST-m6A/ for prediction and reproducibility purposes.

Mutational signature analysis gained significant attention for providing critical insights into the underlying mutational processes for various DNA single base substitution (SBS) signatures and their associations with different cancer types. Recently, RNA single base substitution (RNA-SBS) signatures were defined and described by decomposing RNA variants found in non-small cell lung cancer. Through statistical association, they attributed Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide 3A (APOBEC3A) mutagenesis to the RNA-SBS2 signature. Here, we provide the first validation of an RNA-SBS mutational signature by decomposing novel exogenous and endogenous APOBEC3A RNA editing signatures into COSMICv3.4 RNA-SBS reference signatures. Additionally, we have identified novel RNA-SBS signatures for APOBEC1, APOBEC3B, and APOBEC3G.

AVPs, or antiviral peptides, are short chains of amino acids capable of inhibiting viral replication, preventing viral entry, or disrupting viral membranes. They represent a promising area of research for developing new antiviral therapies due to their potential to target a broad spectrum of viruses, incorporating those resistant to traditional antiviral drugs. However, traditional experimental methods for identifying AVPs are often costly and labour-intensive. Thus far, multiple computational methods have been introduced for the in silico identification of AVPs, but these methods still have certain shortcomings. In this study, we propose a novel stacked ensemble learning framework, termed Stack-AVP, for fast and accurate AVP identification. In Stack-AVP, we investigated heterogeneous prediction models, which were trained with 12 commonly used machine learning algorithms coupled with a wide range of multiple feature encoding schemes. Subsequently, these prediction models were adopted to generate multi-view features providing class information and probability information. Finally, we applied our feature selection method to determine the best feature subset for the construction of the final stacked model. Comparative assessments on the independent test dataset revealed that Stack-AVP surpassed the performance of current state-of-the-art methods, with an accuracy of 0.930, MCC of 0.860, and AUC of 0.975. Furthermore, it was found that our multi-view features exhibited a crucial mechanism to improve the prediction performance of AVPs. To facilitate experimental scientists in performing high-throughput identification of AVPs, the prediction sever Stack-AVP is publicly accessible at https://pmlabqsar.pythonanywhere.com/Stack-AVP.

Non-tuberculous mycobacteria (NTM) have emerged as an increasing threat to public health, due to the extreme antibiotic resistance. NADH pyrophosphatase (NudC) was proposed involving in mycobacterial resistance to the first line anti-tubercular drug isoniazid (INH) or its analog ethionamide (ETH), by hydrolyzing their NAD modified active forms (NAD-INH and NAD-ETH). In this study, we performed enzymatic and structural studies on NudC from M. abscessus (NudC), which is highly resistant to isoniazid and emerging as the most worrisome NTM. We determined the crystal structures of NudC in apo form, substrate NAD-bound form and product AMP-bound form. We observed the mode for the Nudix motif of NudC capturing the pyrophosphate group of NAD mediated by three divalent cation ions, which provides details for understanding the mechanism on NudC hydrolyzing NAD(H) or NAD-capped substrate. Interestingly, our structures revealed a novel subclass NudC from mycobacteria characterized by a unique arginine residue on the conserved QPWPFPxS motif, as well as a unique tower domain that replaces a well-defined zinc-binding motif in E.coli NudC and catalytic domain of mammalian Nudt12. Thus, our structural studies on NudC not only present a class of zinc independent NADH pyrophosphatase in mycobacteria, but also may facilitate the design of NudC inhibitors for the treatment of mycobacteria infections in combination with INH or ETH.

Aminoacyl-tRNA synthetases (ARSs) are responsible for the ligation of amino acids to their cognate tRNAs. In human, nine ARSs form a multi-tRNA synthetase complex (MSC) with three ARS-interacting multifunctional proteins (AIMPs). Among the components of MSC, arginyl-tRNA synthetase 1 (RARS1) and two AIMPs (AIMP1 and AIMP2) have leucine zipper (LZ) motifs, which they utilize for their assembly in an MSC. RARS1 and AIMP1 have two LZ motifs (LZ1 and LZ2) in their N-terminus, respectively, while AIMP2 has one LZ motif between its lysyl-tRNA synthetase 1 (KARS1)-binding motif and glutathione transferase-homology domain, which links aspartyl-tRNA synthetase 1 (DARS1). Although the interaction mode between AIMP1 and RARS1, which also binds glutaminyl-tRNA synthetase 1 (QARS1), has been revealed, the mode in the presence of AIMP2 is still ambiguous since AIMP2 is known to not only bind to AIMP1 but also form a homodimer through its LZ. Here, we determined a crystal structure of the LZ complex of AIMP1 and AIMP2 and revealed the interaction mode of a heterotrimeric complex of RARS1, AIMP1, and AIMP2. The complex is established by a three-stranded coiled-coil structure with RARS1 LZ1, AIMP1 LZ1, and AIMP2 LZ and is completed with a two-stranded coiled-coil structure of RARS1 LZ2 and AIMP1 LZ2. In the human MSC, this heterotrimeric complex of RARS1, AIMP1, and AIMP2 allows for a subcomplex of fourteen protein molecules, in which two QARS1-RARS1-AIMP1-AIMP2-2×KARS1 complexes are linked separately to a dimeric DARS1.

The Gag protein of retroviruses is the primary driver of virus particle assembly. Particle morphologies among retroviral genera are distinct, with intriguing differences observed relative to HIV-1, particularly that of human T-cell leukemia virus type 1 (HTLV-1). In contrast to HIV-1 and other retroviruses where the capsid (CA) carboxy-terminal domain (CTD) possesses the key amino acid determinants involved in driving Gag-Gag interactions, we have previously demonstrated that the amino-terminal domain (NTD) encodes the key residues crucial for Gag multimerization and immature particle production. Here in this study, we sought to thoroughly interrogate the conserved HTLV-1 major homology region (MHR) of the CA to determine whether this region harbors residues important for particle assembly. In particular, site-directed mutagenesis of the HTLV-1 MHR was conducted, and mutants were analyzed for their ability to impact Gag subcellular distribution, particle production and morphology, as well as the CA-CA assembly kinetics. Several key residues (i.e., Q138, E142, Y144, F147 and R150), were found to significantly impact Gag multimerization and particle assembly. Taken together, these observations imply that while the HTLV-1 CA acts as the major region involved in CA-CA interactions, residues in the MHR can impact Gag multimerization, particle assembly and morphology, and likely play an important role in the conformation the CA that is required for CA-CA interactions.

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

In plants, RNA interference (RNAi) serves as a critical defense mechanism against viral infections by regulating gene expression. However, viruses have developed RNA silencing suppressor (RSS) proteins to evade this defense mechanism. The High Plains wheat mosaic virus (HPWMoV) is responsible for the High Plains disease in wheat and produces P7 and P8 proteins, which act as RNA silencing suppressors. P8, in particular, lacks sequence similarity to known suppressors, prompting inquiries into its structure and function. Here, we present a comprehensive analysis of P8, elucidating its structure and function. Using X-ray crystallography, we resolved the full-length P8 structure at 1.9 Å resolution, revealing a tetrameric arrangement formed by two identical dimers. Through structure-based mutagenesis, biochemical assays, and functional studies in plants, we demonstrate that HPWMoV P8's RNA silencing suppression activity relies on its oligomeric state. Contrary to previous report, our findings indicate that while a P8 fused to maltose-binding protein (MBP-P8) was hypothesized to bind short double-stranded RNA, the native P8 tetramer does not interact with small interfering RNA (siRNA). This suggests an alternative mechanism for its function, yet to be determined. Our study sheds light on the structural and functional characteristics of HPWMoV P8, providing valuable insights into the complex interplay between viral suppressors and host defense mechanisms. SIGNIFICANCE STATEMENT Effective action to address malnutrition in all its forms requires an understanding of the mechanisms affecting it. Wheat, crucial for human and animal consumption, faces threats from biotic and abiotic stresses. RNA silencing is a key defense against viral infections in plants. Plant viruses employ various mechanisms, including encoding viral RNA silencing suppression (VRS) proteins, to evade host immune responses. Despite the conservation of RNA-silencing pathways, viral RSS proteins exhibit diverse sequences, structures, and mechanisms. Our study focuses on P8, an RSS protein from HPWMoV. Understanding its structure and assembly is a crucial step toward comprehending how these viruses counteract host defenses, aiding in combatting malnutrition.

Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5' leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including EIF5, IFRD1, MDM2, MIEF1, PPP1R15B, TAF7, and UCP2.

Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.

The apicoplast DNA polymerase (apPol) from Plasmodium falciparum is essential for the parasite's survival, making it a prime target for antimalarial therapies. Here, we present cryo-electron microscopy structures of the apPol in complex with DNA and incoming nucleotide, offering insights into its molecular mechanisms. Our structural analysis reveals that apPol contains critical residues for high-fidelity DNA synthesis, but lacks certain structural elements to confer processive DNA synthesis during replication, suggesting the presence of additional accessory factors. The enzyme exhibits large-scale conformational changes upon DNA and nucleotide binding, particularly within the fingers and thumb subdomains. These movements reveal potential allosteric sites that could serve as targets for drug design. Our findings provide a foundation for advancing the understanding of apPol's unique functional mechanisms and potentially offering new avenues for the development of novel inhibitors and therapeutic interventions against malaria.

Microbiome research has increasingly underscored the profound link between microbial compositions and human health, with numerous studies establishing a strong correlation between microbiome characteristics and various diseases. However, the analysis of microbiome data is frequently compromised by inherent sparsity issues, characterized by a substantial presence of observed zeros. These zeros not only skew the abundance distribution of microbial species but also undermine the reliability of scientific conclusions drawn from such data. Addressing this challenge, we introduce GEMimp, an innovative imputation method designed to infuse robustness into microbiome data analysis. GEMimp leverages the node2vec algorithm, which incorporates both Breadth-First Search (BFS) and Depth-First Search (DFS) strategies in its random walks sampling process. This approach enables GEMimp to learn nuanced, low-dimensional representations of each taxonomic unit, facilitating the reconstruction of their similarity networks with unprecedented accuracy. Our comparative analysis pits GEMimp against state-of-the-art imputation methods including SAVER, MAGIC and mbImpute. The results unequivocally demonstrate that GEMimp outperforms its counterparts by achieving the highest Pearson correlation coefficient when compared to the original raw dataset. Furthermore, GEMimp shows notable proficiency in identifying significant taxa, enhancing the detection of disease-related taxa and effectively mitigating the impact of sparsity on both simulated and real-world datasets, such as those pertaining to Type 2 Diabetes (T2D) and Colorectal Cancer (CRC). These findings collectively highlight the strong effectiveness of GEMimp, allowing for better analysis on microbial data. With alleviation of sparsity issues, it could be greatly facilitated in downstream analyses and even in the field of microbiology.

The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes. The TSC is a key regulator of mTORC1 and is composed of TSC1, TSC2 and TBC1D7. We show a direct interaction of TSC1 with the PIH phospho-binding domain of PIH1D1, which is, surprisingly, phosphorylation independent. Via the use of mutants and KO cell lines, we observe that TSC2 makes independent interactions with HSP90 and the TPR domains of RPAP3. Moreover, inactivation of PIH1D1 or the RUVBL1/2 ATPase activity inhibits the association of TSC1 with TSC2. Taken together, these data suggest a model in which the R2TP recruits TSC1 via PIH1D1 and TSC2 via RPAP3 and HSP90, and use the chaperone-like activities of RUVBL1/2 to stimulate their assembly.

The prefoldin complex is a heterohexameric, evolutionarily conserved co-chaperone that assists in folding of polypeptides downstream of the protein translation machinery. Loss of prefoldin function leads to impaired solubility of cellular proteins. The degradation of proteins by the proteasome is an integral part of protein homeostasis. Failure of regulated protein degradation can lead to the accumulation of misfolded and defective proteins. We show that prefoldin subunit 5 is required for proteasome activity by contributing to the assembly of the 26S proteasome. In particular, we found that absence of the prefoldin subunit 5 impairs formation of the Rpt ring subcomplex of the proteasome. Concomitant deletion of PFD5 and HSM3, a chaperone for assembly of the ATPase subunits comprising the Rpt ring, exacerbates this effect, suggesting a synergistic relationship between the two factors in proteasome assembly. Thus, our findings reveal a regulatory mechanism wherein prefoldin subunit 5 plays a crucial role in maintaining proteasome integrity, thereby influencing the degradation of proteins.

Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (V) and portions of constant (C) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or V domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an IGLV6-57 germline precursor. Comparison of proteins' secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local vs. global conformational stability. Analysis of IgGs vs. corresponding FLCs using hydrogen-deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the C domain. In all IgGs vs. FLCs, segments containing β-strand and α-helix βA-αAB were more dynamic/exposed while segment βD-βE was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose in vivo cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βA-αAB of FLC or βD-βE of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities C-C in FLC and/or C-C in IgG, is a potential therapeutic strategy to complement current approaches targeting V-V or V-C stabilization of LC dimer.

Cytidine (C) to Uridine (U) RNA editing is a post-transcription modification that is involved in diverse biological processes. APOBEC1 (A1) catalyzes the conversion of C-to-U in RNA, which is important in regulating cholesterol metabolism through its editing activity on ApoB mRNA. However, A1 requires a cofactor to form an "editosome" for RNA editing activity. A1CF and RBM47, both RNA-binding proteins, have been identified as cofactors that pair with A1 to form editosomes and edit ApoB mRNA and other cellular RNAs. SYNCRIP is another RNA-binding protein that has been reported as a potential regulator of A1, although it is not directly involved in A1 RNA editing activity. Here, we describe the identification and characterization of a novel cofactor, RBM46 (RNA-Binding-Motif-protein-46), that can facilitate A1 to perform C-to-U editing on ApoB mRNA. Additionally, using the low-error circular RNA sequencing technique, we identified novel cellular RNA targets for the A1/RBM46 editosome. Our findings provide further insight into the complex regulatory network of RNA editing and the potential new function of A1 with its cofactors.

A dozen bHLH-PAS transcription factors have evolved since the dawn of the animal kingdom; nine of them have mutual orthologs between arthropods and vertebrates. These proteins are master regulators in a range of developmental processes from organogenesis, nervous system formation and functioning, to cell fate decisions defining identity of limbs or photoreceptors for color vision. Among the functionally best conserved are bHLH-PAS proteins acting in the animal circadian clock. On the other side of the spectrum are fundamental physiological mechanisms such as those underlying xenobiotic detoxification, oxygen homeostasis, and metabolic adaptation to hypoxia, infection or tumor progression. Predictably, malfunctioning of bHLH-PAS regulators leads to pathologies. Performance of the individual bHLH-PAS proteins is modulated at multiple levels including dimerization and other protein-protein interactions, proteasomal degradation, and by binding low-molecular weight ligands. Despite the vast evolutionary gap dividing arthropods and vertebrates, and the differences in their anatomy, many functions of orthologous bHLH-PAS proteins are remarkably similar, including at the molecular level. Our phylogenetic analysis shows that one bHLH-PAS protein type has been lost during vertebrate evolution. This protein has a unique function as a receptor of the sesquiterpenoid juvenile hormones of insects and crustaceans. Although some other bHLH-PAS proteins are regulated by binding small molecules, the juvenile hormone receptor presents an unprecedented case, since all other non-peptide animal hormones activate members of the nuclear receptor family. The purpose of this review is to compare and highlight parallels and differences in functioning of bHLH-PAS proteins between insects and vertebrates.