Amyloid deposition of the neuroendocrine peptide amylin in islet tissues is a hallmark of type 2 diabetes (T2DM), leading to beta cell toxicity through nutrient deprivation, membrane rupture and apoptosis. Though accumulation of toxic amylin aggregates in islet matrices is well documented, the role of the islet extracellular matrix in mediating amylin aggregation and its pathological consequences remains elusive. Here, we address this question by probing amylin interaction with collagen I (Col I) whose expression in the islet tissue increases during diabetes progression. By combining multiple biophysical techniques, we show that hydrophobic, hydrophilic & cation-pi interactions regulate amylin binding to Col I, with fibrillar collagen driving faster amylin aggregation. Amylin-entangled Col I matrices containing high amounts of amylin induce death and loss of function of INS1E beta-cells. Together, our results illustrate how amylin incorporation in islet matrices through amylin-Col interactions drives T2DM progression by impacting beta-cell viability and insulin secretion.
淀粉样沉积在胰岛组织中的神经内分泌肽淀粉样蛋白是2型糖尿病（T2DM）的标志，它通过营养匮乏、膜破裂和细胞凋亡导致β细胞中毒。虽然胰岛基质中有毒淀粉样蛋白聚集体的积累已被充分证实，但胰岛细胞外基质在介导淀粉样蛋白聚集及其病理后果中的作用仍然难以捉摸。在这里，我们通过探究淀粉样蛋白与胶原蛋白 I（Col I）的相互作用来解决这个问题，胶原蛋白 I 在胰岛组织中的表达随着糖尿病的进展而增加。通过结合多种生物物理技术，我们发现疏水性、亲水性& 阳离子-pi相互作用调节着淀粉蛋白与Col I的结合，而纤维状胶原蛋白会加速淀粉蛋白的聚集。与淀粉蛋白缠结的 Col I 基质含有大量淀粉蛋白，可诱导 INS1E β 细胞死亡并丧失功能。总之，我们的研究结果说明了淀粉蛋白在胰岛基质中的结合是如何通过淀粉蛋白与Col的相互作用影响β细胞的活力和胰岛素分泌，从而推动T2DM的发展。
{"title":"Faster amylin aggregation on fibrillar collagen hastens diabetic progression through β cell death and loss of function","authors":"Md Asrafuddoza Hazari, Gautam Kannan, Akash Kumar Jha, Musale Krushna Pavan, Subrata Dasgupta, Farhin Sultana, Soumya Ranjan Pujahari, Simran Singh, Sarbajeet Dutta, Sai Prasad Pydi, Sankhadeep Dutta, Prasenjit Bhaumik, Hamim Zafar, Ashutosh Kumar, Shamik Sen","doi":"10.1101/2024.08.10.607320","DOIUrl":"https://doi.org/10.1101/2024.08.10.607320","url":null,"abstract":"Amyloid deposition of the neuroendocrine peptide amylin in islet tissues is a hallmark of type 2 diabetes (T2DM), leading to beta cell toxicity through nutrient deprivation, membrane rupture and apoptosis. Though accumulation of toxic amylin aggregates in islet matrices is well documented, the role of the islet extracellular matrix in mediating amylin aggregation and its pathological consequences remains elusive. Here, we address this question by probing amylin interaction with collagen I (Col I) whose expression in the islet tissue increases during diabetes progression. By combining multiple biophysical techniques, we show that hydrophobic, hydrophilic & cation-pi interactions regulate amylin binding to Col I, with fibrillar collagen driving faster amylin aggregation. Amylin-entangled Col I matrices containing high amounts of amylin induce death and loss of function of INS1E beta-cells. Together, our results illustrate how amylin incorporation in islet matrices through amylin-Col interactions drives T2DM progression by impacting beta-cell viability and insulin secretion.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-11DOI: 10.1101/2024.08.10.607472
Bincy Lukose, Saloni Goyal, Athi N Naganathan
H-NS, a nucleoid-associated protein (NAP) from enterobacteria, regulates gene expression by dynamically transducing environmental cues to conformational assembly and DNA binding. In this work, we show that H-NS from Escherichia coli, which can assemble into octameric and tetrameric oligomerization states, forms spontaneous micron-sized liquid-like condensates with DNA at sub-physiological concentrations in vitro. The heterotypic condensates are metastable at 298 K, partially solubilizing with time, while still retaining their liquid-like properties. The condensates display UCST-like phase behavior solubilizing at higher temperatures, but with a large decrease in droplet-assembly propensities at 310 K and also at higher ionic strength. Condensate formation can be tuned in a cyclic manner between 298 and 310 K with the extent of reversibility determined by the incubation time, highlighting strong hysteresis. An engineered phospho-mimetic variant (Y61E) of H-NS, which is dimeric and only weakly binds DNA, is unable to form condensates. The Y61E mutant solubilizes pre-formed H-NS condensates with DNA in a few minutes with nearly an order of magnitude speed-up in droplet dissolution at 310 K relative to 298 K, demonstrating rapid molecular transport between dilute and condensed phases. Our results establish that the oligomerization of H-NS is intrinsically tied not only to DNA binding but also its phase-separation tendencies, while showcasing the regulatable and programmable nature of heterotypic condensates formed by an archetypal NAP via multiple cues and their lifetimes.
H-NS是一种来自肠杆菌的核团相关蛋白（NAP），它通过动态地将环境线索传递给构象组装和DNA结合来调节基因表达。在这项研究中，我们发现大肠杆菌的 H-NS 可以组装成八聚体和四聚体的寡聚状态，在体外亚生理浓度下与 DNA 自发形成微米大小的液态凝集物。这种异型凝聚物在 298 K 时是稳定的，随着时间的推移会部分溶解，但仍保持其液态特性。冷凝物显示出类似 UCST 的相行为，在较高温度下会发生溶解，但在 310 K 和较高离子强度下，液滴组装的倾向性会大幅下降。凝结物的形成可在 298 至 310 K 之间以循环方式进行调整，其可逆性程度由培养时间决定，突出显示了很强的滞后性。H-NS的一个工程磷酸化模拟变体（Y61E）是二聚体，只能与DNA微弱结合，无法形成凝结物。Y61E 突变体能在几分钟内溶解与 DNA 预先形成的 H-NS 凝聚物，在 310 K 温度下，液滴溶解速度比 298 K 温度下加快了近一个数量级，这表明稀释相和凝聚相之间的分子运输非常迅速。我们的研究结果证明，H-NS 的寡聚化不仅与 DNA 结合有内在联系，而且还与其相分离倾向有关，同时还展示了由典型 NAP 通过多种线索及其生命周期形成的异型凝聚物的可调节性和可编程性。
{"title":"Oligomerization-Mediated Phase-Separation in the Nucleoid-Associated Sensory Protein H-NS is Controlled by Ambient Cues","authors":"Bincy Lukose, Saloni Goyal, Athi N Naganathan","doi":"10.1101/2024.08.10.607472","DOIUrl":"https://doi.org/10.1101/2024.08.10.607472","url":null,"abstract":"H-NS, a nucleoid-associated protein (NAP) from enterobacteria, regulates gene expression by dynamically transducing environmental cues to conformational assembly and DNA binding. In this work, we show that H-NS from Escherichia coli, which can assemble into octameric and tetrameric oligomerization states, forms spontaneous micron-sized liquid-like condensates with DNA at sub-physiological concentrations in vitro. The heterotypic condensates are metastable at 298 K, partially solubilizing with time, while still retaining their liquid-like properties. The condensates display UCST-like phase behavior solubilizing at higher temperatures, but with a large decrease in droplet-assembly propensities at 310 K and also at higher ionic strength. Condensate formation can be tuned in a cyclic manner between 298 and 310 K with the extent of reversibility determined by the incubation time, highlighting strong hysteresis. An engineered phospho-mimetic variant (Y61E) of H-NS, which is dimeric and only weakly binds DNA, is unable to form condensates. The Y61E mutant solubilizes pre-formed H-NS condensates with DNA in a few minutes with nearly an order of magnitude speed-up in droplet dissolution at 310 K relative to 298 K, demonstrating rapid molecular transport between dilute and condensed phases. Our results establish that the oligomerization of H-NS is intrinsically tied not only to DNA binding but also its phase-separation tendencies, while showcasing the regulatable and programmable nature of heterotypic condensates formed by an archetypal NAP via multiple cues and their lifetimes.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607354
Zheyao Hu, Jordi Marti
The NRAS-mutant subset of melanoma is one of the most aggressive and lethal types associated with poor overall survival. Unfortunately, a low understanding of the NRAS-mutant dynamic behavior has lead to the lack of clinically approved therapeutic agents able to directly target NRAS oncogenes. In this work, accurate local structures of NRAS and its mutants have been fully explored through the corresponding free energy surfaces obtained by microsecond scale well-tempered metadynamics simulations. Free energy calculations are crucial to reveal the precise mechanisms of Q61 mutations at the atomic level. Considering specific atom-atom distances d and angles φ as appropriate reaction coordinates we have obtained free energy surfaces revealing local and global minima together with their main transitions states, unvealing the mechanisms of abnormal NRAS activation from atomic-level and quantitatively analyzing the corresponding stable states. This will help to advance in our understanding of the basic mechanisms of NRAS mutations, offering new opportunities for the design of potential inhibitors.
{"title":"Quantitative comparison of the structural differences between NRAS and its mutations by well-tempered metadynamics simulations","authors":"Zheyao Hu, Jordi Marti","doi":"10.1101/2024.08.09.607354","DOIUrl":"https://doi.org/10.1101/2024.08.09.607354","url":null,"abstract":"The NRAS-mutant subset of melanoma is one of the most aggressive and lethal types associated with poor overall survival. Unfortunately, a low understanding of the NRAS-mutant dynamic behavior has lead to the lack of clinically approved therapeutic agents able to directly target NRAS oncogenes. In this work, accurate local structures of NRAS and its mutants have been fully explored through the corresponding free energy surfaces obtained by microsecond scale well-tempered metadynamics simulations. Free energy calculations are crucial to reveal the precise mechanisms of Q61 mutations at the atomic level. Considering specific atom-atom distances d and angles φ as appropriate reaction coordinates we have obtained free energy surfaces revealing local and global minima together with their main transitions states, unvealing the mechanisms of abnormal NRAS activation from atomic-level and quantitatively analyzing the corresponding stable states. This will help to advance in our understanding of the basic mechanisms of NRAS mutations, offering new opportunities for the design of potential inhibitors.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607387
Uttam Anand, Shubhadeep Patra, Rohith Vedhthaanth Sekar, Craig R Garen, Michael T Woodside
Misfolding of the protein PrP causes prion diseases in mammals. Disease susceptibility varies widely among species, despite PrP sequences differing by only a few amino acids. How these differences alter PrP folding and misfolding remains unclear. We compared the folding dynamics of single PrP molecules from three species with different disease susceptibility: dogs (immune), hamsters (susceptible), and bank voles (extremely susceptible). Measurements with optical tweezers revealed important differences between the folding cooperativity, pathways, energy barriers, and kinetics of these proteins. In contrast to the two-state folding of hamster PrP, dog PrP always folded through multiple intermediates. However, both featured rapid native folding, homogeneous energy barriers, and no readily observable misfolding. Bank vole PrP also folded via intermediates, but more slowly and via inhomogeneous barriers. Most notably, it formed several metastable misfolded states starting from the unfolded state. Analyzing the sequence of intermediates seen in pulling curves, we found significant differences in the folding pathways for dog and bank vole PrP, implying that sequence mutations altered energy barriers so as to redirect folding pathways. These results show that subtle differences in PrP sequence between species produce profound changes in folding behavior, providing insight into the factors underlying misfolding propensity.
{"title":"Different folding mechanisms in prion proteins from mammals with different disease susceptibility observed at the single-molecule level","authors":"Uttam Anand, Shubhadeep Patra, Rohith Vedhthaanth Sekar, Craig R Garen, Michael T Woodside","doi":"10.1101/2024.08.09.607387","DOIUrl":"https://doi.org/10.1101/2024.08.09.607387","url":null,"abstract":"Misfolding of the protein PrP causes prion diseases in mammals. Disease susceptibility varies widely among species, despite PrP sequences differing by only a few amino acids. How these differences alter PrP folding and misfolding remains unclear. We compared the folding dynamics of single PrP molecules from three species with different disease susceptibility: dogs (immune), hamsters (susceptible), and bank voles (extremely susceptible). Measurements with optical tweezers revealed important differences between the folding cooperativity, pathways, energy barriers, and kinetics of these proteins. In contrast to the two-state folding of hamster PrP, dog PrP always folded through multiple intermediates. However, both featured rapid native folding, homogeneous energy barriers, and no readily observable misfolding. Bank vole PrP also folded via intermediates, but more slowly and via inhomogeneous barriers. Most notably, it formed several metastable misfolded states starting from the unfolded state. Analyzing the sequence of intermediates seen in pulling curves, we found significant differences in the folding pathways for dog and bank vole PrP, implying that sequence mutations altered energy barriers so as to redirect folding pathways. These results show that subtle differences in PrP sequence between species produce profound changes in folding behavior, providing insight into the factors underlying misfolding propensity.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"45 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Biomolecular condensates formed via liquid-liquid phase separation are ubiquitous in cells, especially in the nucleus. While condensates containing one or two kinds of biomolecules have been relatively well characterized, those with more hetero-genous biomolecular components and interactions between biomolecules inside are largely unknown. This study used resi-due-resolution molecular dynamics simulations to investigate heterogeneous protein assemblies that include four master tran-scription factors in mammalian embryonic stem cells: Oct4, Sox2, Klf4, and Nanog. Simulations of the mixture systems showed highly heterogeneous and dynamic behaviors; the condensates mainly contained Sox2, Klf4, and Nanog, while Oct4 was dissolved into the dilute phase. Condensates consisted of loosely interacting clusters in which Klf4 was the most abun-dant in the cores. We suggest that Klf4 serves as a scaffold of the condensate where Sox2 and Nanog are bound to stabilize the condensate, whereas Oct4 is moderately recruited to the condensate, serving as a client mainly via its interaction with Sox2.
{"title":"Heterogenous organization in condensates of multiple transcription factors in embryonic stem cells","authors":"Azuki Mizutani, Cheng Tan, Yuji Sugita, Shoji Takada","doi":"10.1101/2024.06.14.599027","DOIUrl":"https://doi.org/10.1101/2024.06.14.599027","url":null,"abstract":"Biomolecular condensates formed via liquid-liquid phase separation are ubiquitous in cells, especially in the nucleus. While condensates containing one or two kinds of biomolecules have been relatively well characterized, those with more hetero-genous biomolecular components and interactions between biomolecules inside are largely unknown. This study used resi-due-resolution molecular dynamics simulations to investigate heterogeneous protein assemblies that include four master tran-scription factors in mammalian embryonic stem cells: Oct4, Sox2, Klf4, and Nanog. Simulations of the mixture systems showed highly heterogeneous and dynamic behaviors; the condensates mainly contained Sox2, Klf4, and Nanog, while Oct4 was dissolved into the dilute phase. Condensates consisted of loosely interacting clusters in which Klf4 was the most abun-dant in the cores. We suggest that Klf4 serves as a scaffold of the condensate where Sox2 and Nanog are bound to stabilize the condensate, whereas Oct4 is moderately recruited to the condensate, serving as a client mainly via its interaction with Sox2.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607375
Christopher Bergevin, Rebecca Whiley, Hero Wit, Geoffrey Manley, Pim van Dijk
As a sound pressure detector that uses energy to boost both its sensitivity and selectivity, the inner ear is an active non-equilibrium system. The collective processes of the inner ear giving rise to this exquisite functionality remain poorly understood. One manifestation of the active ear across the animal kingdom is the presence of spontaneous otoacoustic emission (SOAE), idiosyncratic arrays of spectral peaks that can be measured using a sensitive microphone in the ear canal.1 Current SOAE models attempt to explain how multiple peaks arise, and generally assume a spatially-distributed tonotopic system. However, the nature of the generators, their coupling, and the role of noise (e.g., Brownian motion) are hotly debated, especially given the inner ear morphological diversity across vertebrates. One means of probing these facets of emission generation is studying fluctuations in SOAE peak properties, which produce amplitude (AM) and frequency modulations (FM). These properties are likely related to the presence of noise affecting active cellular generation elements, and the coupling between generators. To better biophysically constrain models, this study characterizes the fluctuations in filtered SOAE peak waveforms, focusing on interrelations within and across peaks. A systematic approach is taken, examining three species that exhibit disparate inner ear morphologies: humans, barn owls, and green anole lizards. To varying degrees across all three groups, SOAE peaks have intra- (IrP) and interpeak (IPP) correlations indicative of interactions between generative elements. Activity from anole lizards, whose auditory sensory organ is relatively much smaller than that of humans or barn owls, showed a much higher incidence of IPP correlations. Taken together, we propose that these data are indicative of SOAE cellular generators acting cooperatively, allowing the ear to function as an optimized detector.
{"title":"Auditory Cellular Cooperativity Probed Via Spontaneous Otoacoustic Emissions","authors":"Christopher Bergevin, Rebecca Whiley, Hero Wit, Geoffrey Manley, Pim van Dijk","doi":"10.1101/2024.08.09.607375","DOIUrl":"https://doi.org/10.1101/2024.08.09.607375","url":null,"abstract":"As a sound pressure detector that uses energy to boost both its sensitivity and selectivity, the inner ear is an active non-equilibrium system. The collective processes of the inner ear giving rise to this exquisite functionality remain poorly understood. One manifestation of the active ear across the animal kingdom is the presence of spontaneous otoacoustic emission (SOAE), idiosyncratic arrays of spectral peaks that can be measured using a sensitive microphone in the ear canal.1 Current SOAE models attempt to explain how multiple peaks arise, and generally assume a spatially-distributed tonotopic system. However, the nature of the generators, their coupling, and the role of noise (e.g., Brownian motion) are hotly debated, especially given the inner ear morphological diversity across vertebrates. One means of probing these facets of emission generation is studying fluctuations in SOAE peak properties, which produce amplitude (AM) and frequency modulations (FM). These properties are likely related to the presence of noise affecting active cellular generation elements, and the coupling between generators. To better biophysically constrain models, this study characterizes the fluctuations in filtered SOAE peak waveforms, focusing on interrelations within and across peaks. A systematic approach is taken, examining three species that exhibit disparate inner ear morphologies: humans, barn owls, and green anole lizards. To varying degrees across all three groups, SOAE peaks have intra- (IrP) and interpeak (IPP) correlations indicative of interactions between generative elements. Activity from anole lizards, whose auditory sensory organ is relatively much smaller than that of humans or barn owls, showed a much higher incidence of IPP correlations. Taken together, we propose that these data are indicative of SOAE cellular generators acting cooperatively, allowing the ear to function as an optimized detector.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"84 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945059","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607385
Richard J Lindsay, Rafael Giordano Viegas, VITOR B P LEITE, Philip Anthony Wigge, Sonya M Hanson
The evening complex (EC) is a tripartite DNA repressor and a core component of the circadian clock that provides a mechanism for temperature-responsive growth and development of many plants. ELF3, a component of the EC, is a disordered scaffolding protein that blocks transcription of growth genes at low temperature. At increased temperature EC DNA binding is disrupted and ELF3 is sequestered in a reversible nuclear condensate, allowing transcription and growth to proceed. The condensation is driven by a low complexity prion-like domain (PrD), and the sensitivity of the temperature response is modulated by the length of a variable polyQ tract, with a longer polyQ tract corresponding to enhanced condensate formation and hypocotyl growth at increased temperature. Here, a series of computational studies provides evidence that polyQ tracts promote formation of temperature-sensitive helices in flanking residues with potential impacts for EC stability under increasing temperature. REST2 simulations uncover a heat-induced population of condensation-prone conformations that results from the exposure of 'sticky' aromatic residues by temperature-responsive breaking of long-range contacts. Coarse-grained Martini simulations reveal both polyQ tract length and sequence context modulate the temperature dependence of cluster formation. Understanding the molecular mechanism underlying the ELF3-PrD temperature response in plants has implications for technologies including modular temperature-response elements for heat-responsive protein design and agricultural advances to enable optimization of crop yields and allow plants to thrive in increasingly inhospitable environments.
黄昏复合体（EC）是一种三方 DNA 抑制因子，也是昼夜节律钟的核心组成部分，它为许多植物的温度响应性生长和发育提供了一种机制。ELF3是EC的一个组成部分，它是一种紊乱的支架蛋白，在低温下会阻碍生长基因的转录。温度升高时，EC 的 DNA 结合被破坏，ELF3 被封闭在可逆的核凝结物中，从而使转录和生长得以继续。凝结是由一个低复杂度的朊病毒样结构域（PrD）驱动的，温度反应的敏感性受可变多Q束长度的调节，较长的多Q束在温度升高时会增强凝结的形成和下胚轴的生长。在此，一系列计算研究提供了证据，证明polyQ束能促进侧翼残基中温度敏感螺旋的形成，从而对温度升高时EC的稳定性产生潜在影响。REST2 模拟发现了由热诱导的易凝结构象群，这是由于 "粘性 "芳香残基暴露于温度反应性长程接触断裂所致。粗粒度马蒂尼模拟显示，多Q束长度和序列上下文都会调节聚类形成的温度依赖性。了解植物中ELF3-PrD温度响应的分子机制对各种技术都有影响，包括用于热响应蛋白质设计的模块化温度响应元件和农业技术进步，从而优化作物产量并使植物在日益恶劣的环境中茁壮成长。
{"title":"Molecular dynamics simulations illuminate the role of sequence context in the ELF3-PrD-based temperature sensing mechanism in plants","authors":"Richard J Lindsay, Rafael Giordano Viegas, VITOR B P LEITE, Philip Anthony Wigge, Sonya M Hanson","doi":"10.1101/2024.08.09.607385","DOIUrl":"https://doi.org/10.1101/2024.08.09.607385","url":null,"abstract":"The evening complex (EC) is a tripartite DNA repressor and a core component of the circadian clock that provides a mechanism for temperature-responsive growth and development of many plants. ELF3, a component of the EC, is a disordered scaffolding protein that blocks transcription of growth genes at low temperature. At increased temperature EC DNA binding is disrupted and ELF3 is sequestered in a reversible nuclear condensate, allowing transcription and growth to proceed. The condensation is driven by a low complexity prion-like domain (PrD), and the sensitivity of the temperature response is modulated by the length of a variable polyQ tract, with a longer polyQ tract corresponding to enhanced condensate formation and hypocotyl growth at increased temperature. Here, a series of computational studies provides evidence that polyQ tracts promote formation of temperature-sensitive helices in flanking residues with potential impacts for EC stability under increasing temperature. REST2 simulations uncover a heat-induced population of condensation-prone conformations that results from the exposure of 'sticky' aromatic residues by temperature-responsive breaking of long-range contacts. Coarse-grained Martini simulations reveal both polyQ tract length and sequence context modulate the temperature dependence of cluster formation. Understanding the molecular mechanism underlying the ELF3-PrD temperature response in plants has implications for technologies including modular temperature-response elements for heat-responsive protein design and agricultural advances to enable optimization of crop yields and allow plants to thrive in increasingly inhospitable environments.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607002
Kira Devantier, Trine L. Toft-Bertelsen, Andreas Prestel, Viktoria M. S. Kjaer, Cagla Sahin, Marco Giulini, Stavroula Louka, Katja Spiess, Asmita Manandhar, Katrine Qvortrup, Trond Ulven, Bo Hjorth Bentzen, Alexandre Bonvin, Nanna MacAulay, Birthe B. Kragelund, Mette M Rosenkilde
Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.
病毒感染呈上升趋势，需要针对病毒蛋白的药物。病毒蛋白是一类不断增加的病毒编码跨膜寡聚蛋白，可使小分子穿过膜。尽管病毒蛋白结构稀少，但最近的研究揭示了跨膜螺旋数量和寡聚状态的多样性。在这里，我们提供了证据，证明腮腺炎病毒的小疏水蛋白（SH）是一种五聚体病毒蛋白。根据大量生物物理数据，全长 SH 的 HADDOCK 模型显示，其细胞内 C 端区域形成了对稳定五聚体至关重要的扩展结构。在爪蟾卵母细胞中异源表达野生型 SH 和变体后，发现 viroporin 是一种氯离子通道，由内衬孔的保守羟基携带残基促进。小分子 BIT225 可抑制 SH 的通道功能，突出了通过 SH 进行抗病毒靶向的潜力。
{"title":"The SH Protein of Mumps Virus is a Druggable Pentameric Viroporin","authors":"Kira Devantier, Trine L. Toft-Bertelsen, Andreas Prestel, Viktoria M. S. Kjaer, Cagla Sahin, Marco Giulini, Stavroula Louka, Katja Spiess, Asmita Manandhar, Katrine Qvortrup, Trond Ulven, Bo Hjorth Bentzen, Alexandre Bonvin, Nanna MacAulay, Birthe B. Kragelund, Mette M Rosenkilde","doi":"10.1101/2024.08.09.607002","DOIUrl":"https://doi.org/10.1101/2024.08.09.607002","url":null,"abstract":"Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607107
Jingtian Xu, Yong Wang
Understanding and predicting the diverse conformational states of membrane proteins is essential for elucidating their biological functions. Despite advancements in computational methods, accurately capturing these complex structural changes remains a significant challenge. In this study, we introduce a method for predicting diverse functional states of membrane protein conformations using a diffusion model. Our approach integrates forward and backward diffusion processes, incorporating state classifiers and additional conditioners to control the generation gradient of conformational states. We specifically target the P-type ATPases, a key membrane transporter, for which we curated and expanded a structural dataset. By employing a graph neural network with a custom membrane constraint, our model generates precise structures for P-type ATPases across different functional states. This approach represents a significant step forward in computational structural biology and holds great potential for studying the dynamics of other membrane proteins.
了解和预测膜蛋白的各种构象状态对于阐明其生物功能至关重要。尽管计算方法不断进步，但准确捕捉这些复杂的结构变化仍是一项重大挑战。在本研究中，我们介绍了一种利用扩散模型预测膜蛋白构象的不同功能状态的方法。我们的方法整合了前向和后向扩散过程，纳入了状态分类器和附加调节器，以控制构象状态的生成梯度。我们特别以 P 型 ATP 酶（一种关键的膜转运体）为研究对象，并对其结构数据集进行了整理和扩充。通过使用带有自定义膜约束的图神经网络，我们的模型生成了 P 型 ATPases 在不同功能状态下的精确结构。这种方法标志着计算结构生物学向前迈出了重要一步，并为研究其他膜蛋白的动力学提供了巨大潜力。
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Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607004
Alessio Accardi, Sayan Chakraborty, Zhang Feng, Sangyun Lee, Omar E Alvarenga, Aniruddha Panda, Renato Bruni, George Khelashvili, Kallol Gupta
Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create an electronegative surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.
{"title":"Structure and function of the human apoptotic scramblase Xkr4","authors":"Alessio Accardi, Sayan Chakraborty, Zhang Feng, Sangyun Lee, Omar E Alvarenga, Aniruddha Panda, Renato Bruni, George Khelashvili, Kallol Gupta","doi":"10.1101/2024.08.07.607004","DOIUrl":"https://doi.org/10.1101/2024.08.07.607004","url":null,"abstract":"Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create an electronegative surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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