Journal of Molecular Biology最新文献

UvrD-family Superfamily 1A helicases are processive ATP-dependent motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD-family monomers translocate along single stranded (ss) DNA with 3'-to-5' directionality but must be activated by dimerization to become helicases in the absence of force or accessory factors. Mycobacterium tuberculosis (Mtb) UvrD1 helicase forms dimers via a disulfide bond between native cysteines in the 2B sub-domains of each monomer. E. coli UvrD forms non-covalent dimers using the same 2B domain interface as in Mtb UvrD1. Using both ensemble and single DNA molecule approaches we examined an E. coli UvrD variant (R421C), which forms covalent dimers with constitutive helicase activity. For the first time this has enabled us to compare the ssDNA translocation and helicase activities of covalent dimers and monomers. Crosslinked UvrD dimers exhibit much higher ssDNA translocation processivities than monomers, although with similar translocation rates. Crosslinked UvrD dimers also show highly processive DNA unwinding of thousands of base pairs, much higher than non-crosslinked UvrD dimers, while monomers show no DNA unwinding activity. DNA unwinding rates of crosslinked UvrD dimers are only ∼20% slower than ssDNA translocation rates, indicating they are "active" helicases that directly facilitate duplex destabilization.

Understanding and manipulating the 3D structure and dynamics of molecular systems is central to all molecular sciences including structural biology. MolecularWebXR's new HandMol module provides a browser-based, multiuser environment for immersive molecular visualization and interactive modeling across all modern devices. Leveraging the WebXR standard, HandMol requires no installation, allowing users to seamlessly access shared virtual rooms from VR/AR headsets, mobile devices, or computers. HandMol enables natural interaction through hand tracking, allowing users to query atom information by pointing or to manipulate structures using bare-hand gestures. Real-time physics feedback is provided via integrated engines, including ANI-2x for small molecules, AMBER14 for atomistic biomolecular systems, and CALVADOS coarse-grained models. We demonstrate HandMol's utility through 3D-difficult tasks like ligand (un)docking, driving conformational changes, and preparing starting poses for simulations. This globally accessible platform facilitates scientific discussion, hypothesis testing, and immersive education by bridging high-performance molecular dynamics with intuitive, natural control in a collaborative virtual space. Resource link:https://molecularwebxr.org/ Direct link to HandMol:https://molecularwebxr.org/create.

Type II topoisomerases are found in all kingdoms of life and perform key biological functions during replication, transcription, and other DNA metabolic events. These enzymes are also the targets of important classes of anti-bacterial and anti-cancer drugs. Clinically active drugs targeting topoisomerase II primarily act as poisons rather than as enzyme inhibitors. During the normal enzymatic reaction, topoisomerase poisons stabilize the normally reversible covalent enzyme/DNA adduct leading to DNA damage. More recently, Top2 mutants have been identified that exert the same effect as these small molecules, and expression of these mutants in cells leads to genome instability. Mutations that increase the potency of topoisomerase targeting drugs have the potential to mimic drug action and expand insight into the drug mechanism. Here we carried out an extensive screen of mutations in human Top2α to identify mutations that result in elevated levels of sensitivity to the anti-cancer drug etoposide. This screen was conducted by expressing a library of mutagenized human Top2α that complemented a yeast temperature sensitive Top2 allele in rad52- yeast strains. Etoposide hypersensitive mutations were identified throughout the catalytic domains of Top2α. Mutants were typically hypersensitive to other Top2 inhibitors, although some mutants had hypersensitivity that was specific for etoposide. Several examples of mutant proteins identified in the screen were purified and characterized biochemically. These proteins exhibited a broad range of alterations in enzyme activity including altered requirements for divalent cations, altered relaxation and decatenation activity, altered utilization of ATP, and enhanced DNA cleavage in the absence of inhibitors. Our results suggest that diverse mechanisms influence the ability of small molecules to convert type II topoisomerases to DNA damaging agents, and the etoposide hypersensitivity mutations found provide landmarks for the development of small molecules with improved potency.

The growing availability of 3D protein structures enables large-scale assessment of missense variants and their phenotypic impact. Missense3D, first developed in 2019, predicts the structural consequences of amino acid substitutions and provides mechanistic explanations by identifying features such as cavities, steric clashes and disrupted non-covalent bonds that may cause protein misfolding. The new Missense3D algorithm presented here accepts genomics, protein, and structural coordinates; models variants on both single proteins and protein-protein complexes using experimental and predicted structures, including AlphaFold models; performs automated sequence-structure mapping; and evaluates structural model quality before analysis. The Missense3D web server is freely available to all users, including commercial ones, at https://missense3d.bc.ic.ac.uk/~missense3d2/.

MAGI1 is a multidomain scaffold protein that coordinates signaling complexes involved in cell adhesion, polarity, and signal transduction. While PDZ domains are generally regarded as independent interaction modules, the role of interdomain communication in regulating partner recognition remains poorly understood. Here, we investigated the molecular basis of partner recognition by its sixth PDZ domain (PDZ6) and the contribution of neighboring domains to this process. Using stopped-flow kinetics, we characterized the interactions of PDZ6 with two physiological partners, β-catenin and TRIP6. The two proteins displayed distinct kinetic and thermodynamic behaviors, indicating different recognition mechanisms despite targeting the same domain. Analysis of ionic strength and pH dependence revealed distinct molecular determinants underlying the two interactions. While β-catenin binding was largely insensitive to the multidomain context, TRIP6 recognition was selectively influenced by adjacent domains. Remarkably, binding of DLL1 to the neighboring PDZ5 domain strongly enhanced TRIP6 affinity while exerting minimal effects on β-catenin, and this effect was further modulated by pH. Together, these findings support a model in which MAGI1 behaves as a dynamically coupled scaffold whose binding properties are shaped by interdomain communication, highlighting the importance of studying PDZ-mediated interactions within their multidomain context.

Lactate and histone H3K18 lactylation (H3K18la) have been linked to glioblastoma biology. We examined their relationship with synaptotagmin 8 (SYT8) and the response to temozolomide (TMZ). U251MG and U87MG cells were cultured under normoxia or hypoxia and exposed to TMZ, sodium lactate or both. H3K18la enrichment at the SYT8 promoter was assessed by chromatin immunoprecipitation. The effects of SYT8 knockdown were evaluated using CCK-8, colony formation, flow cytometry, wound healing and Transwell assays. Lactate and protein abundance were measured, including H3K18la normalized to total H3 in the four-group experiment. Endpoint tumor weight was assessed in a subcutaneous U87MG xenograft model. TMZ reduced CCK-8 signal and intracellular lactate in both cell lines, whereas lactate supplementation attenuated several effects of TMZ. H3K18la was enriched at the SYT8 promoter, and SYT8 knockdown was accompanied by lower lactate and H3K18la signals. Under TMZ treatment, SYT8-depleted cells formed fewer colonies and had a higher apoptosis fraction. Lactate partly reversed these changes and increased H3K18la/total H3 while SYT8 protein remained low. The combined SYT8-knockdown and TMZ group had the lowest endpoint tumor weight. These results link SYT8 to lactate-associated H3K18 lactylation and TMZ response in GBM cells. The reciprocal changes in SYT8, lactate and H3K18la suggest a possible feedback relationship that warrants further mechanistic study.

Ribosome biogenesis is a highly coordinated pathway that involves the assembly of ribosomal RNAs (rRNAs) with ribosomal proteins (r-proteins) to generate functional ribosomal subunits (r-subunits). The Saccharomyces cerevisiae (yeast) large 60S r-subunit consists of three rRNA molecules and 46 r-proteins. The contributions of nearly all r-proteins of the yeast large r-subunit have been characterized; however, a few non-essential proteins remain poorly understood. Although non-essential, human eL22 has been identified as a key player in p53 regulation during ribosomal stress and as a highly mutated target in cancers. Despite this function, the role of eL22 in ribosome maturation is still ill-defined. In this study, we characterized yeast eL22 r-protein, which is encoded by two paralogous genes that exhibit markedly different expression levels. Our results show that eL22 assembles into intermediate nucleolar pre-60S ribosomal particles. Loss of eL22 impairs cell growth and reduces 60S r-subunit accumulation, phenotypes that are exacerbated at low temperatures. Analysis of pre-rRNA processing by pulse-chase labeling, northern blot hybridization, and primer extension reveals a defect in 27S pre-rRNA maturation, specifically at the level of 27SB pre-rRNA processing. Consequently, nuclear export of eL22-deficient pre-60S particles is mildly impaired. Furthermore, we identify genetic interactions between eL22 and neighboring r-proteins, eL38 and eL31. We conclude that eL22 assembly is required for optimal pre-60S maturation during middle nucleolar stages, particularly at low temperatures, a function likely supported by the cooperative action of other r-proteins associated with common elements of 25S rRNA.

When evolution removes a residue essential for catalysis, the protein fold may survive, but whether the former active site simply decays or is physicochemically remodeled remains unclear. We explored this question in the HEPN family of ribonucleases. HEPN proteins normally cleave RNA using a conserved R-X(4-6)-H motif, in which histidine plays a central catalytic role. Using the structure-first workflow now implemented as FOLDTRACE, we searched the AlphaFold/UniProt50 representative database for HEPN-fold proteins without requiring retention of the catalytic motif. Among the 1,000 top-ranked hits, 836 lacked an explicit HEPN name or attached HEPN-clan Pfam cross-reference. Of these, 750 were DUF86/UPF0331 proteins already recognizable as HEPN-related at the family level, illustrating incomplete per-record cross-referencing rather than discovery of an unknown HEPN family. Eighty-six proteins lacked the canonical catalytic histidine, and most retained the ancestral HEPN cleft. Phylogenetic analyses showed repeated H/non-H state changes. Monomer-level electrostatic screening identified a subset with strong negative shifts, but dimer reconstruction showed why these calls require oligomeric validation: A0A852UQU6 retained a strong shift at the composite active site, whereas another monomer-level candidate did not. Together, these results establish recurrent catalytic-state change and physicochemical remodeling of the ancestral HEPN cleft. Whether such remodeling corresponds to altered binding, regulation, or another molecular function remains a hypothesis requiring experimental testing.

Glycosylation is implicated in many diseases, from cancer to neurodegeneration. Elucidating these disease mechanisms requires precise identification of glycosylation sites. Computational prediction provides an efficient complement to labor-intensive experiments, yet further accuracy improvements are needed for practical use. Here, we present LoRA-N-Gly, a lightweight and efficient end-to-end approach that leverages Low-Rank Adaptation (LoRA) to fine-tune the ESM2-3B protein language model for direct, residue-level prediction of N-linked glycosylation sites. By updating only a small set of LoRA parameters, LoRA-N-Gly substantially reduces trainable parameters and training cost while remaining sequence-only (protein sequence as the sole input feature). LoRA-N-Gly, which maps raw sequences directly to site probabilities without handcrafted features or separate post-processing stages, achieves the best performance among the methods compared on predicting N-linked glycosylation sites.

A server-based program, DC+, is presented for analysis of residual dipolar couplings (RDCs) and residual chemical shift anisotropy (RCSA) in proteins and nucleic acids. The program aims to meet commonly encountered needs in the analysis of RDCs. In addition to standard fitting of RDCs to known internuclear vector orientations using standard singular value decomposition (SVD), a multitude of features allow adjustment of such fits to specific cases: (1) fitting to the sum of RDCs, such as commonly measured for methylene or methyl ¹³C-¹H vectors, (2) fitting of RDCs to ensembles of structures, with either equal or optimized weights for the ensemble members, (3) fitting of RDCs to idealized α-helical or canonical nucleic-acid duplex structure, (4) orthogonalization of multiple sets of alignment tensors and generation of orthogonalized RDC sets, and (5) fitting of RCSA to either a static structure or an ensemble. The program includes options to weigh experimental RDCs by the inverse of their reported uncertainty, to select different sets of RDCs for validation from those used for fitting, and to carry out jackknifed cross-validation, a feature that is particularly useful when only few RDCs (< ∼25) are available. DC+ also contains a robust non-linear least-squares fitting mode, suitable for evaluation of data in cases where the magnitude and/or asymmetry of the alignment tensor are known. By integrating fitting, validation, uncertainty analysis, ensemble optimization, and visualization within a single web interface, DC+ provides a comprehensive environment for routine and advanced RDC analysis. The server is freely available at https://spin.niddk.nih.gov/bax-apps/nmrserver/dc+/.