Pub Date : 2024-10-18DOI: 10.1016/j.jmb.2024.168823
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.
折叠中间体既介导蛋白质折叠，也介导包括肌萎缩性脊髓侧索硬化症（ALS）在内的人类疾病中观察到的错误折叠和聚集，是治疗干预的主要目标。在这项研究中，我们采用实验和计算相结合的方法，在与 ALS 相关的 RNA 结合蛋白 TDP-43 的第二个 RNA 识别基序（RRM2）中确定了折叠中间体的核心结构核。尿素平衡展开研究发现，RRM2 中间状态由坍塌的残余二级结构组成，这些二级结构位于 RRM2 的 N 端半部分，而 C 端大部分处于无序状态。这些研究结果表明，RRM2 中间体在正常的 TDP-43 功能中发挥作用，并通过低稳定性和非原生二级结构成为错误折叠和聚集的模板。
{"title":"A Hydrophobic Core Stabilizes the Residual Structure in the RRM2 Intermediate State of the ALS-linked Protein TDP-43","authors":"","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":null,"pages":null},"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}
Pub Date : 2024-10-18DOI: 10.1016/j.jmb.2024.168819
Eliot Courtney, Amitava Datta, David H Mathews, Max Ward
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-10-17DOI: 10.1016/j.jmb.2024.168821
Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. Vitis vinifera 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.
脂氧合酶催化膜脂中游离或酯化的多不饱和脂肪酸链发生过氧化反应。葡萄 LoxA 在葡萄成熟时转录诱导，定位于叶绿体内膜，负责半乳糖脂特异性单过氧化和二过氧化。在这里，我们介绍了 LoxA 的动力学和结构特征。我们的 X 射线结构揭示了一个组成型二聚体，其构象在洗涤剂的诱导下发生了变化，从而影响了底物结合和催化作用。在封闭构象中，一个 LID 结构域通过立体阻碍阻止底物进入催化位点。洗涤剂添加到 CMC 以上会破坏 LID 的稳定性，并打开二聚体，使两个催化位点都能从 PLAT 结构域框定的同一表面进入。因此，洗涤剂分子占据了 PLAT/催化结构域界面上的异构位点。在胶束中提供底物时，酶活性和正合作性都会增加，从而反映出这些结构变化。通过色氨酸荧光评估，当定点突变使二聚体不稳定时，与胶束相互作用的能力就会丧失。根据我们的数据，可以提出一个膜上蛋白质活化模型，将 LoxA 归类为一种直接从膜上作用于脂肪酸链的界面酶，类似于哺乳动物的 15-LOX 和 5-LOX。
{"title":"Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces","authors":"","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":null,"pages":null},"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}
Pub Date : 2024-10-09DOI: 10.1016/j.jmb.2024.168818
Trevor Norton, Debswapna Bhattacharya
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.
{"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":null,"pages":null},"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}
Pub Date : 2024-10-09DOI: 10.1016/j.jmb.2024.168815
Nucleotide incorporation and lacZ-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 in vitro-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 in vitro-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.
{"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":"","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 Mg<sup>2+</sup>) using an <em>in vitro</em>-transcribed RNA were around 7.3 × 10<sup>−5</sup>. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg<sup>2+</sup> 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<sup>−5</sup>). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg<sup>2+</sup> (7.4 × 10<sup>−5</sup>) were similar to those determined with the <em>in vitro</em>-transcribed RNA, and were reduced to 1.8 × 10<sup>−5</sup> in the presence of 0.5 mM Mg<sup>2+</sup>. 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":null,"pages":null},"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}
Pub Date : 2024-10-09DOI: 10.1016/j.jmb.2024.168816
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.
{"title":"Structure Prediction of Large RNAs with AlphaFold3 Highlights its Capabilities and Limitations","authors":"","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":null,"pages":null},"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}
Pub Date : 2024-10-05DOI: 10.1016/j.jmb.2024.168813
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.
在哺乳动物细胞中，DNA 连接酶 1（LIG1）是基因组复制和单链断裂修复的主要 DNA 连接酶。据报道，人类 LIG1 的几个突变（包括 R305Q、R641L 和 R771W）会导致原发性免疫缺陷 LIG1 综合征。R641L 和 R771W 突变分别位于核苷酸转移酶结构域和寡核苷酸结合结构域，已被生物化学鉴定并证明会降低催化效率，而最近报道的 DNA 结合结构域（DBD）内的 R305Q 突变在机理上仍未得到探讨。众所周知，R641L 和 R771W 突变会在催化过程中影响结构域间的相互作用和 DNA 结合，从而降低 LIG1 的催化活性，但不会显著影响 DNA 的总体亲和力。为了阐明导致 LIG1 综合征的 R305Q 突变的分子基础，我们纯化了这种单残基突变蛋白，并研究了其二级结构、蛋白稳定性、DNA 结合亲和力和催化效率。我们的研究结果表明，R305Q突变通过破坏DBD-DNA相互作用，显著损害了LIG1的功能，导致LIG1的DNA结合亲和力降低了7至21倍，催化效率降低了33至300倍。此外，R305Q 突变会使 LIG1 蛋白的稳定性略微降低 2 到 3.6 °C，与之前观察到的 R641L 或 R771W 突变体的效果相当。总之，我们的研究结果发现了一种新的机制，即 R305Q 突变会损害 LIG1 催化的缺口 DNA 连接，导致 LIG1 综合征，并强调了 DBD-DNA 相互作用在紧密的 DNA 结合和高效的 LIG1 催化中的关键作用。
{"title":"Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease","authors":"","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":null,"pages":null},"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}
Pub Date : 2024-10-05DOI: 10.1016/j.jmb.2024.168814
Rachel A Mooney, Junqiao Zhu, Jason Saba, Robert Landick
The accurate and efficient biogenesis of RNA by cellular RNA polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, and termination of transcription. Of the many discovered to date, the elongation regulator NusG-Spt5 is the only universally conserved transcription factor. With orthologs and paralogs found in all three domains of life, this ubiquity underscores their ancient and essential regulatory functions. NusG-Spt5 proteins evolved to maintain a similar binding interface to RNAP through contacts of the NusG N-terminal domain (NGN) that bridge the main DNA-binding cleft. We propose that varying strength of these contacts, modulated by tethering interactions, either decrease transcriptional pausing by smoothing the rugged thermodynamic landscape of transcript elongation or enhance pausing, depending on which conformation of RNAP is stabilized by NGN contacts. NusG-Spt5 contains one (in bacteria and archaea) or more (in eukaryotes) C-terminal domains that use a KOW fold to contact diverse targets, tether the NGN, and control RNA biogenesis. Recent work highlights these diverse functions in different organisms. Some bacteria contain multiple specialized NusG paralogs that regulate subsets of operons via sequence-specific targeting, controlling production of antibiotics, toxins, or capsule proteins. Despite their common origin, NusG orthologs can differ in their target selection, interacting partners, and effects on RNA synthesis. We describe the current understanding of NusG-Spt5 structure, interactions with RNAP and other regulators, and cellular functions including significant recent progress from genome-wide analyses, single-molecule visualization, and cryo-EM. The recent findings highlight the remarkable diversity of function among these structurally conserved proteins.
{"title":"NusG-Spt5 Transcription Factors: Universal, Dynamic Modulators of Gene Expression.","authors":"Rachel A Mooney, Junqiao Zhu, Jason Saba, Robert Landick","doi":"10.1016/j.jmb.2024.168814","DOIUrl":"10.1016/j.jmb.2024.168814","url":null,"abstract":"<p><p>The accurate and efficient biogenesis of RNA by cellular RNA polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, and termination of transcription. Of the many discovered to date, the elongation regulator NusG-Spt5 is the only universally conserved transcription factor. With orthologs and paralogs found in all three domains of life, this ubiquity underscores their ancient and essential regulatory functions. NusG-Spt5 proteins evolved to maintain a similar binding interface to RNAP through contacts of the NusG N-terminal domain (NGN) that bridge the main DNA-binding cleft. We propose that varying strength of these contacts, modulated by tethering interactions, either decrease transcriptional pausing by smoothing the rugged thermodynamic landscape of transcript elongation or enhance pausing, depending on which conformation of RNAP is stabilized by NGN contacts. NusG-Spt5 contains one (in bacteria and archaea) or more (in eukaryotes) C-terminal domains that use a KOW fold to contact diverse targets, tether the NGN, and control RNA biogenesis. Recent work highlights these diverse functions in different organisms. Some bacteria contain multiple specialized NusG paralogs that regulate subsets of operons via sequence-specific targeting, controlling production of antibiotics, toxins, or capsule proteins. Despite their common origin, NusG orthologs can differ in their target selection, interacting partners, and effects on RNA synthesis. We describe the current understanding of NusG-Spt5 structure, interactions with RNAP and other regulators, and cellular functions including significant recent progress from genome-wide analyses, single-molecule visualization, and cryo-EM. The recent findings highlight the remarkable diversity of function among these structurally conserved proteins.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":null,"pages":null},"PeriodicalIF":4.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142387012","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}
Pub Date : 2024-10-05DOI: 10.1016/j.jmb.2024.168817
{"title":"From space vaccinology to molecular mechanisms of choice, vaccinology needs molecular biology","authors":"","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":null,"pages":null},"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}
Pub Date : 2024-10-05DOI: 10.1016/j.jmb.2024.168811
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.
{"title":"Molecular Basis of the Recognition of the Active Rab8a by Optineurin","authors":"","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":null,"pages":null},"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}
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