Journal of Molecular Biology最新文献

{"title":"A Hydrophobic Core Stabilizes the Residual Structure in the RRM2 Intermediate State of the ALS-linked Protein TDP-43","authors":"Brian C. Mackness ,&nbsp;Brittany R. Morgan ,&nbsp;Laura M. Deveau ,&nbsp;Sagar V. Kathuria ,&nbsp;Jill A. Zitzewitz ,&nbsp;Francesca Massi","doi":"10.1016/j.jmb.2024.168823","DOIUrl":"10.1016/j.jmb.2024.168823","url":null,"abstract":"<div><div>Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43 (TAR DNA-binding protein-43), using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168823"},"PeriodicalIF":4.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"memerna: Sparse RNA Folding Including Coaxial Stacking.","authors":"Eliot Courtney, Amitava Datta, David H Mathews, Max Ward","doi":"10.1016/j.jmb.2024.168819","DOIUrl":"https://doi.org/10.1016/j.jmb.2024.168819","url":null,"abstract":"<p><p>Determining RNA secondary structure is a core problem in computational biology. Fast algorithms for predicting secondary structure are fundamental to this task.Department of Biochemistry & Biophysics, University of Rochester Medical Center, Rochester, NY, USA We describe a modified formulation of the Zuker-Stiegler algorithm with coaxial stacking, a stabilising interaction in which the ends of helices in multi-loops are stacked. In particular, optimal coaxial stacking is computed as part of the dynamic programming state, rather than in an inner loop. We introduce a new notion of sparsity, which we call replaceability. Replaceability is a more general condition and applicable in more places than the triangle inequality that is used by previous sparse folding methods. We also introduce non-monotonic candidate lists as an additional sparsification tool. Existing usages of the triangle inequality for sparsification can be thought of as an application of both replaceability and monotonicity together. The modified recurrences along with replaceability allows sparsification to be applied to coaxial stacking as well, which increases the speed of the algorithm. We implemented this algorithm in software we call memerna, which we show to have the fastest exact (non-heuristic) implementation of RNA folding under the complete Turner 2004 model with coaxial stacking, out of several popular RNA folding tools supporting coaxial stacking. We also introduce a new notation for secondary structure which includes coaxial stacking, terminal mismatches, and dangles (CTDs) information. The memerna package 0.1 release is available at https://github.com/Edgeworth/memerna/tree/release/0.1.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168819"},"PeriodicalIF":4.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces","authors":"Stefania Pilati ,&nbsp;Klemens Wild ,&nbsp;Andrea Gumiero ,&nbsp;Iris Holdermann ,&nbsp;Yvonne Hackmann ,&nbsp;Mauro Dalla Serra ,&nbsp;Graziano Guella ,&nbsp;Claudio Moser ,&nbsp;Irmgard Sinning","doi":"10.1016/j.jmb.2024.168821","DOIUrl":"10.1016/j.jmb.2024.168821","url":null,"abstract":"<div><div>Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. <em>Vitis vinifera</em> LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplast membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interface. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168821"},"PeriodicalIF":4.7,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142454994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sifting through the noise: A survey of diffusion probabilistic models and their applications to biomolecules.","authors":"Trevor Norton, Debswapna Bhattacharya","doi":"10.1016/j.jmb.2024.168818","DOIUrl":"10.1016/j.jmb.2024.168818","url":null,"abstract":"<p><p>Diffusion probabilistic models have made their way into a number of high-profile applications since their inception. In particular, there has been a wave of research into using diffusion models in the prediction and design of biomolecular structures and sequences. Their growing ubiquity makes it imperative for researchers in these fields to understand them. This paper serves as a general overview for the theory behind these models and the current state of research. We first introduce diffusion models and discuss common motifs used when applying them to biomolecules. We then present the significant outcomes achieved through the application of these models in generative and predictive tasks. This survey aims to provide readers with a comprehensive understanding of the increasingly critical role of diffusion models.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168818"},"PeriodicalIF":4.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells","authors":"Javier Martínez del Río ,&nbsp;Estrella Frutos-Beltrán ,&nbsp;Alba Sebastián-Martín ,&nbsp;Fátima Lasala ,&nbsp;Kiyoshi Yasukawa ,&nbsp;Rafael Delgado ,&nbsp;Luis Menéndez-Arias","doi":"10.1016/j.jmb.2024.168815","DOIUrl":"10.1016/j.jmb.2024.168815","url":null,"abstract":"<div><div>Nucleotide incorporation and <em>lacZ</em>-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg²⁺) using an <em>in vitro</em>-transcribed RNA were around 7.3 × 10⁻⁵. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg²⁺ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10⁻⁵). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg²⁺ (7.4 × 10⁻⁵) were similar to those determined with the <em>in vitro</em>-transcribed RNA, and were reduced to 1.8 × 10⁻⁵ in the presence of 0.5 mM Mg²⁺. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168815"},"PeriodicalIF":4.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure Prediction of Large RNAs with AlphaFold3 Highlights its Capabilities and Limitations","authors":"Robert T. McDonnell,&nbsp;Aaron N. Henderson,&nbsp;Adrian H. Elcock","doi":"10.1016/j.jmb.2024.168816","DOIUrl":"10.1016/j.jmb.2024.168816","url":null,"abstract":"<div><div>DeepMind’s AlphaFold3 webserver offers exciting new opportunities to make structural predictions of heterogeneous macromolecular systems. Here we attempt to apply AlphaFold3 to large RNA molecules whose 3D atomic structures are unknown but whose physical dimensions have been studied experimentally. One difficulty that we encounter is that models returned by AlphaFold3 often contain severe steric clashes and, less frequently, clear breaks in the phosphodiester backbone, with the probability of both events increasing with the length of the RNA. Restricting attention to those RNAs for which non-clashing models can be obtained, we find that hydrodynamic radii computed from the AlphaFold3 models are much larger than those reported experimentally under low salt conditions but are in better agreement with those reported in the presence of polyvalent cations. For two RNAs whose shapes have been imaged experimentally, the computed anisotropies of the AlphaFold3-predicted structures are too low, indicating that they are excessively spherical; extending this analysis to larger RNAs shows that they become progressively more spherical with increasing length. Overall, the results suggest that AlphaFold3 is capable of producing plausible models for RNAs up to ∼2000 nucleotides in length, but that thousands of predictions may be required to obtain models free of geometric problems.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168816"},"PeriodicalIF":4.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease","authors":"Nikita Zalenski,&nbsp;Yufan He,&nbsp;Zucai Suo","doi":"10.1016/j.jmb.2024.168813","DOIUrl":"10.1016/j.jmb.2024.168813","url":null,"abstract":"<div><div>In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7–21-fold lower DNA binding affinity and a 33–300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1’s protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168813"},"PeriodicalIF":4.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From space vaccinology to molecular mechanisms of choice, vaccinology needs molecular biology","authors":"Tobias R. Kollmann,&nbsp;Manish Sadarangani,&nbsp;Richard B. Kennedy","doi":"10.1016/j.jmb.2024.168817","DOIUrl":"10.1016/j.jmb.2024.168817","url":null,"abstract":"","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168817"},"PeriodicalIF":4.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142379792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Basis of the Recognition of the Active Rab8a by Optineurin","authors":"Jing Zhang ,&nbsp;Lei Liu ,&nbsp;Miao Li ,&nbsp;Haobo Liu ,&nbsp;Xinyu Gong ,&nbsp;Yubin Tang ,&nbsp;Yuchao Zhang ,&nbsp;Xindi Zhou ,&nbsp;Zhiqiao Lin ,&nbsp;Hanbo Guo ,&nbsp;Lifeng Pan","doi":"10.1016/j.jmb.2024.168811","DOIUrl":"10.1016/j.jmb.2024.168811","url":null,"abstract":"<div><div>Optineurin (OPTN), a multifunctional adaptor protein in mammals, plays critical roles in many cellular processes, such as vesicular trafficking and autophagy. Notably, mutations in optineurin are directly associated with many human diseases, such as amyotrophic lateral sclerosis (ALS). OPTN can specifically recognize Rab8a and the GTPase-activating protein TBC1D17, and facilitate the inactivation of Rab8a mediated by TBC1D17, but with poorly understood mechanism. Here, using biochemical and structural approaches, we systematically characterize the interaction between OPTN and Rab8a, revealing that OPTN selectively recognizes the GTP-bound active Rab8a through its leucine-zipper domain (LZD). The determined crystal structure of OPTN LZD in complex with the active Rab8a not only elucidates the detailed binding mechanism of OPTN with Rab8a but also uncovers a unique binding mode of Rab8a with its effectors. Furthermore, we demonstrate that the central coiled-coil domain of OPTN and the active Rab8a can simultaneously interact with the TBC domain of TBC1D17 to form a ternary complex. Finally, based on the OPTN LZD/Rab8a complex structure and relevant biochemical analyses, we also evaluate several known ALS-associated mutations found in the LZD of OPTN. Collectively, our findings provide mechanistic insights into the interaction of OPTN with Rab8a, expanding our understanding of the binding modes of Rab8a with its effectors and the potential etiology of diseases caused by OPTN mutations.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 22","pages":"Article 168811"},"PeriodicalIF":4.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the Protein Universe","authors":"Louise C. Serpell,&nbsp;Daniel E. Otzen,&nbsp;Sheena E. Radford","doi":"10.1016/j.jmb.2024.168812","DOIUrl":"10.1016/j.jmb.2024.168812","url":null,"abstract":"","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 21","pages":"Article 168812"},"PeriodicalIF":4.7,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378938","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}