Pub Date : 2024-09-05DOI: 10.1101/2024.09.04.611325
Nitesh K Khandelwal, Meghna Gupta, James E. Gomez, Sulyman Barkho, Ziqiang Guan, Ashley Y. Eng, Tomo Kawate, Sree Ganesh Balasubramani, Andrej Sali, Deborah T. Hung, Robert M. Stroud
A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 - 3.4 Å resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for alternate access to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the alternate access mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.
我们对结核分枝杆菌（Mtb）进行了广泛的化学遗传学筛选，以确定已确定或以前未开发的治疗开发靶点的抑制剂，结果发现了抑制重要外排泵 EfpA 的化合物（BRD-8000.3 和 BRD-9327）。为了了解这些化合物的抑制机制，我们以 2.7 - 3.4 Å 的分辨率测定了与抑制剂结合的 EfpA 的结构。我们的结构揭示了两种抑制剂的不同抑制机制。BRD-8000.3 结合在与脂质双分子层接触的隧道中，并向中心空腔延伸，从而取代了结合在 apo 结构中的脂质分子的脂肪酸链，这表明它阻断了天然脂质底物的进入途径，而它对小分子底物溴化乙锭的抑制机制则是非竞争性的，后者可能是通过另一条隧道进入的。与此同时，BRD-9327 与外前庭结合，并没有完全阻断底物通向外部的路径，这表明它可能抑制了交替进入膜的两个不同侧面所需的动态运动，这也是主要促进剂超家族（MFS）转运体的特征。这两种抑制剂都可能在抑制交替进入机制方面发挥作用，从而解释了它们对某些底物的非竞争性流出。我们的研究结果解释了这些抑制剂协同作用的基础，以及它们在抗结核治疗的多药策略中的组合潜力。它们还潜在地指出了这种重要的外排泵作为脂质转运体的可能功能。这些结构为合理改造这些抑制剂以提高药效奠定了基础。
{"title":"Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A","authors":"Nitesh K Khandelwal, Meghna Gupta, James E. Gomez, Sulyman Barkho, Ziqiang Guan, Ashley Y. Eng, Tomo Kawate, Sree Ganesh Balasubramani, Andrej Sali, Deborah T. Hung, Robert M. Stroud","doi":"10.1101/2024.09.04.611325","DOIUrl":"https://doi.org/10.1101/2024.09.04.611325","url":null,"abstract":"A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 - 3.4 Å resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for alternate access to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the alternate access mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176097","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 : 2024-09-05DOI: 10.1101/2024.09.05.611543
John S Allingham, Daria Trofimova, Caitlin Doubleday, Byron Hunter, Jesus Danilo Serrano Arevalo, Emma Davison, Eric Wen, Kim Munro
Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal αβ-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of CaKip3 folds into a compact 92 Å-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.
驱动蛋白-8 运动蛋白在有丝分裂过程中通过调节着丝点连接的微管的动态以及有丝分裂纺锤体的长度和位置，帮助染色体的排列和分离。一些驱动蛋白-8 同工型通过作为多功能机械酶来完成这些作用，它们可以穿过微管，聚集在微管的加端，然后移除末端 αβ-tubulin 亚基。虽然这些活动主要由运动结构域驱动，其独特的结构-功能关系最近已有报道，但非运动尾部结构域含有尚未从结构上阐明的整体功能元素。以白念珠菌 Kip3 蛋白为驱动蛋白-8 的模型系统，我们展示了一种 X 射线晶体结构和流体力学数据，显示了尾部的马达结构域-近端区段如何指导两个驱动蛋白-8 多肽组装成一个同源二聚体，从而形成该马达的柄。与大多数其他运动驱动蛋白柄的延长盘卷形成螺旋不同，CaKip3 的近端尾部折叠成一个紧凑的 92 Å 长的四螺旋束，并形成二聚体。第一和第三螺旋为二聚体界面提供了大部分表面积，而另外两个螺旋则支撑着折叠的柄结构。螺旋束的上部和下部由一个柔性铰链隔开，当弯曲时，柄的外表面形状略有不同。我们认为，这些独特的特征为驱动蛋白-8 的柄提供了结构刚度，同时也提供了与驱动蛋白-8 相关蛋白或其他马达调控区域进行瞬时相互作用的场所。
{"title":"Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle","authors":"John S Allingham, Daria Trofimova, Caitlin Doubleday, Byron Hunter, Jesus Danilo Serrano Arevalo, Emma Davison, Eric Wen, Kim Munro","doi":"10.1101/2024.09.05.611543","DOIUrl":"https://doi.org/10.1101/2024.09.05.611543","url":null,"abstract":"Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal αβ-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the <em>Candida albicans</em> Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of <em>Ca</em>Kip3 folds into a compact 92 Å-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176091","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 : 2024-09-05DOI: 10.1101/2024.09.03.608162
Andrey G Baranovskiy, Lucia M Morstadt, Nigar D Babayeva, Tahir H. Tahirov
The human primosome, a four-subunit complex of DNA primase and DNA polymerase alpha (Polalpha), plays a critical role in DNA replication by initiating RNA and DNA synthesis on both chromosome strands. A recent study has shown that a major virulence factor in the SARS-CoV-2 infection, Nsp1 (non structural protein 1), forms a stable complex with Polalpha but does not affect the primosome activity. Here we show that Nsp1 inhibits DNA synthesis across inverted repeats prone to hairpin formation. Analysis of current structural data revealed the overlapping binding sites for Nsp1 and the winged helix-turn-helix domain of RPA (wHTH) on Polalpha, indicating a competition between them. Comparison of the inhibitory effect of Nsp1 and wHTH on DNA hairpin bypass by Polalpha showed an 8-fold lower IC50 value for Nsp1 (1 uM). This study provides a valuable insight into the mechanism of inhibition of human DNA replication by Nsp1 during a SARS-CoV-2 infection.
人类底物小体是由 DNA 底物酶和 DNA 聚合酶α（Polalpha）组成的四亚基复合物，在 DNA 复制过程中起着关键作用，可启动两条染色体链上的 RNA 和 DNA 合成。最近的一项研究表明，SARS-CoV-2 感染中的一个主要毒力因子 Nsp1（非结构蛋白 1）与 Polalpha 形成稳定的复合物，但不影响底物酶的活性。在这里，我们发现 Nsp1 可抑制容易形成发夹的倒置重复序列上的 DNA 合成。对现有结构数据的分析表明，Nsp1 与 Polalpha 上 RPA 的翼螺旋-转螺旋结构域（wHTH）的结合位点重叠，表明它们之间存在竞争。比较了 Nsp1 和 wHTH 对 Polalpha 绕过 DNA 发夹的抑制作用，结果表明 Nsp1 的 IC50 值（1 uM）比 wHTH 低 8 倍。这项研究为了解 Nsp1 在 SARS-CoV-2 感染期间抑制人类 DNA 复制的机制提供了宝贵的见解。
{"title":"Nsp1 stalls DNA Polymerase alpha at DNA hairpins","authors":"Andrey G Baranovskiy, Lucia M Morstadt, Nigar D Babayeva, Tahir H. Tahirov","doi":"10.1101/2024.09.03.608162","DOIUrl":"https://doi.org/10.1101/2024.09.03.608162","url":null,"abstract":"The human primosome, a four-subunit complex of DNA primase and DNA polymerase alpha (Polalpha), plays a critical role in DNA replication by initiating RNA and DNA synthesis on both chromosome strands. A recent study has shown that a major virulence factor in the SARS-CoV-2 infection, Nsp1 (non structural protein 1), forms a stable complex with Polalpha but does not affect the primosome activity. Here we show that Nsp1 inhibits DNA synthesis across inverted repeats prone to hairpin formation. Analysis of current structural data revealed the overlapping binding sites for Nsp1 and the winged helix-turn-helix domain of RPA (wHTH) on Polalpha, indicating a competition between them. Comparison of the inhibitory effect of Nsp1 and wHTH on DNA hairpin bypass by Polalpha showed an 8-fold lower IC50 value for Nsp1 (1 uM). This study provides a valuable insight into the mechanism of inhibition of human DNA replication by Nsp1 during a SARS-CoV-2 infection.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176092","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 : 2024-09-04DOI: 10.1101/2024.09.04.611255
Yueming Long, Ariane Mora, Emre Guersoy, Kadina E. Johnston, Francesca-Zhoufan Li, Frances H. Arnold
Sequence-function data provides valuable information about the protein functional landscape, but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq's ability to accurately detect variants under various experimental conditions. Finally, we show LevSeq's utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.
{"title":"LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning","authors":"Yueming Long, Ariane Mora, Emre Guersoy, Kadina E. Johnston, Francesca-Zhoufan Li, Frances H. Arnold","doi":"10.1101/2024.09.04.611255","DOIUrl":"https://doi.org/10.1101/2024.09.04.611255","url":null,"abstract":"Sequence-function data provides valuable information about the protein functional landscape, but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq's ability to accurately detect variants under various experimental conditions. Finally, we show LevSeq's utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176094","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 : 2024-09-04DOI: 10.1101/2024.09.04.611182
Lin Wei, Hai-Yan Cao, Ruyi Zou, Min Du, Qingdong Zhang, Danrong Lu, Xiangyu Xu, Yingying Xu, Wenshuang Wang, Xiu-Lan Chen, Yu-Zhong Zhang, Fuchuan Li
Recently, a new class of glycosaminoglycan (GAG) lyases (GAGases) belonging to PL35 family has been discovered with an ultrabroad substrate spectrum that can degrade three types of uronic acid-containing GAGs (hyaluronic acid, chondroitin sulfate and heparan sulfate) or even alginate. In this study, the structures of GAGase II from Spirosoma fluviale and GAGase VII from Bacteroides intestinalis DSM 17393 were determined at 1.9 and 2.4 Angstrom resolution, respectively, and their catalytic mechanism was investigated by the site-directed mutant of their crucial residues and molecular docking assay. Structural analysis showed that GAGase II and GAGase VII consist of an N-terminal (alpha/alpha)7 toroid multidomain and a C-terminal two-layered beta-sheet domain with Mn2+. Notably, although GAGases share similar folds and catalytic mechanisms with some GAG lyases and alginate lyases, they exhibit higher structural homology with alginate lyases than GAG lyases, which may present a crucial structural evidence for the speculation that GAG lyases with (alpha/alpha)n toroid and antiparallel beta-sheet structures arrived by a divergent evolution from alginate lyases with the same folds. Overall, this study not only solved the structure of PL35 GAG lyases for the first time and investigated their catalytic mechanism, especially the reason why GAGase III can additionally degrade alginate, but also provided a key clue in the divergent evolution of GAG lyases that originated from alginate lyases.
最近发现了一类属于 PL35 家族的新的糖胺聚糖（GAG）裂解酶（GAGase），其底物谱极广，可降解三种含尿酸的 GAG（透明质酸、硫酸软骨素和硫酸肝素）甚至藻酸盐。本研究分别以 1.9 埃和 2.4 埃的分辨率测定了来自 Spirosoma fluviale 的 GAGase II 和来自 Bacteroides intestinalis DSM 17393 的 GAGase VII 的结构，并通过对其关键残基的定点突变和分子对接试验研究了它们的催化机理。结构分析表明，GAGase II 和 GAGase VII 由 N 端（α/α）7 环状多域和 C 端带有 Mn2+ 的两层β片状结构域组成。值得注意的是，虽然 GAG 酶与一些 GAG 裂解酶和藻酸盐裂解酶具有相似的折叠和催化机制，但它们与藻酸盐裂解酶的结构同源性却高于 GAG 裂解酶，这可能为具有（α/α）n 环状结构和反平行 beta 片结构的 GAG 裂解酶是由具有相同折叠的藻酸盐裂解酶分化进化而来的推测提供了重要的结构证据。总之，本研究不仅首次解析了PL35 GAG裂解酶的结构，研究了其催化机理，尤其是GAG酶III能够额外降解藻酸盐的原因，而且为GAG裂解酶起源于藻酸盐裂解酶的分化进化提供了关键线索。
{"title":"Crystal structure and catalytic mechanism of PL35 family glycosaminoglycan lyases with an ultrabroad substrate spectrum","authors":"Lin Wei, Hai-Yan Cao, Ruyi Zou, Min Du, Qingdong Zhang, Danrong Lu, Xiangyu Xu, Yingying Xu, Wenshuang Wang, Xiu-Lan Chen, Yu-Zhong Zhang, Fuchuan Li","doi":"10.1101/2024.09.04.611182","DOIUrl":"https://doi.org/10.1101/2024.09.04.611182","url":null,"abstract":"Recently, a new class of glycosaminoglycan (GAG) lyases (GAGases) belonging to PL35 family has been discovered with an ultrabroad substrate spectrum that can degrade three types of uronic acid-containing GAGs (hyaluronic acid, chondroitin sulfate and heparan sulfate) or even alginate. In this study, the structures of GAGase II from Spirosoma fluviale and GAGase VII from Bacteroides intestinalis DSM 17393 were determined at 1.9 and 2.4 Angstrom resolution, respectively, and their catalytic mechanism was investigated by the site-directed mutant of their crucial residues and molecular docking assay. Structural analysis showed that GAGase II and GAGase VII consist of an N-terminal (alpha/alpha)7 toroid multidomain and a C-terminal two-layered beta-sheet domain with Mn2+. Notably, although GAGases share similar folds and catalytic mechanisms with some GAG lyases and alginate lyases, they exhibit higher structural homology with alginate lyases than GAG lyases, which may present a crucial structural evidence for the speculation that GAG lyases with (alpha/alpha)n toroid and antiparallel beta-sheet structures arrived by a divergent evolution from alginate lyases with the same folds. Overall, this study not only solved the structure of PL35 GAG lyases for the first time and investigated their catalytic mechanism, especially the reason why GAGase III can additionally degrade alginate, but also provided a key clue in the divergent evolution of GAG lyases that originated from alginate lyases.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176098","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 : 2024-09-04DOI: 10.1101/2024.09.04.611235
James Tolchard, Vicki S. Chambers, Laurence S. Boutemy, Mark J Banfield, Tharin M. A. Blumenschein
Oomycete pathogens cause large economic losses in agriculture through diseases such as late blight (Phytophthora infestans), and stem and root rot of soybean (Phytophthora sojae). The effector protein AVR3a, from P. infestans, and its homologue AVR3a11 from P. capsici, are examples of host-translocated effectors that interact with plant proteins to evade defence mechanisms and enable infection. Both proteins belong to the family of RXLR effectors and contain an N-terminal secretion signal, an RXLR motif for translocation into the host cell, and a C-terminal effector domain. Within this family, a large number of proteins have been predicted to contain one or more WY domains as their effector domain, and this domain is proposed to encompass a conserved minimal core fold containing three helices, further stabilised by additional helices or dimerization. In AVR3a11, a helical N-terminal extension to the core fold forms a four-helix bundle, as determined by X-ray crystallography. For a complete picture of the dynamics of AVR3a11, we have determined the solution structure of AVR3a11, and studied its dynamics in the fast timescale (ns-ps, from NMR relaxation parameters) and in the slow timescale (seconds to minutes, from hydrogen/deuterium exchange experiments). Hydrogen/deuterium exchange showed that the N-terminal helix is less stable than the other three helices, confirming the core fold originally proposed. Relaxation measurements confirm that AVR3a11 undergoes extensive conformational exchange, despite the uniform presence of fast motions in the spectral density function throughout most of its sequence. As functional residues are located in the more mobile regions, this flexibility in the slow/intermediate timescale may be functionally important.
卵菌病原体通过晚疫病（Phytophthora infestans）、大豆茎腐病和根腐病（Phytophthora sojae）等病害给农业造成了巨大的经济损失。侵染疫霉菌的效应蛋白 AVR3a 和荚膜疫霉菌的同源蛋白 AVR3a11 是宿主转座效应蛋白的典范，它们与植物蛋白相互作用，躲避防御机制并促成感染。这两种蛋白都属于 RXLR 效应子家族，包含一个 N 端分泌信号、一个用于转位到宿主细胞的 RXLR 基序和一个 C 端效应子结构域。在该家族中，大量蛋白质被预测含有一个或多个 WY 结构域作为其效应结构域，该结构域被认为包含一个保守的最小核心折叠，其中含有三个螺旋，并通过额外的螺旋或二聚化进一步稳定。在 AVR3a11 中，核心折叠的螺旋 N 端延伸形成了一个四螺旋束，这是由 X 射线晶体学确定的。为了全面了解 AVR3a11 的动态，我们确定了 AVR3a11 的溶液结构，并研究了它在快时间尺度（ns-ps，来自核磁共振弛豫参数）和慢时间尺度（秒到分钟，来自氢/氘交换实验）上的动态。氢/氘交换显示，N 端螺旋的稳定性低于其他三个螺旋，这证实了最初提出的核心折叠。弛豫测量证实，尽管 AVR3a11 的频谱密度函数在其大部分序列中一致存在快速运动，但它仍经历了广泛的构象交换。由于功能残基位于移动性较强的区域，这种慢/中时间尺度上的灵活性可能具有重要的功能意义。
{"title":"Dynamics in the Phytophthora capsici effector AVR3a11 confirm the core WY domain fold","authors":"James Tolchard, Vicki S. Chambers, Laurence S. Boutemy, Mark J Banfield, Tharin M. A. Blumenschein","doi":"10.1101/2024.09.04.611235","DOIUrl":"https://doi.org/10.1101/2024.09.04.611235","url":null,"abstract":"Oomycete pathogens cause large economic losses in agriculture through diseases such as late blight (Phytophthora infestans), and stem and root rot of soybean (Phytophthora sojae). The effector protein AVR3a, from P. infestans, and its homologue AVR3a11 from P. capsici, are examples of host-translocated effectors that interact with plant proteins to evade defence mechanisms and enable infection. Both proteins belong to the family of RXLR effectors and contain an N-terminal secretion signal, an RXLR motif for translocation into the host cell, and a C-terminal effector domain. Within this family, a large number of proteins have been predicted to contain one or more WY domains as their effector domain, and this domain is proposed to encompass a conserved minimal core fold containing three helices, further stabilised by additional helices or dimerization. In AVR3a11, a helical N-terminal extension to the core fold forms a four-helix bundle, as determined by X-ray crystallography. For a complete picture of the dynamics of AVR3a11, we have determined the solution structure of AVR3a11, and studied its dynamics in the fast timescale (ns-ps, from NMR relaxation parameters) and in the slow timescale (seconds to minutes, from hydrogen/deuterium exchange experiments). Hydrogen/deuterium exchange showed that the N-terminal helix is less stable than the other three helices, confirming the core fold originally proposed. Relaxation measurements confirm that AVR3a11 undergoes extensive conformational exchange, despite the uniform presence of fast motions in the spectral density function throughout most of its sequence. As functional residues are located in the more mobile regions, this flexibility in the slow/intermediate timescale may be functionally important.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175861","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 : 2024-09-04DOI: 10.1101/2024.09.04.610228
Donghoon Kang, Yeji Lim, Dabin Ahn, Jaeseok Lee, Chin-Ju Park
Cellular senescence, marked by irreversible cell cycle arrest and the secretion of proinflammatory factors, contributes to aging and cancer recurrence. Chemotherapy can also induce senescence; senescent cells often resist apoptosis and promote tumor recurrence. The interaction between FOXO4 and p53 sustains cell survival. Herein, we used biophysical techniques and conducted cellular experiments to develop a peptide inhibitor targeting this interaction to eliminate senescent cancer cells. We identified key regions in the p53 transactivation domain (TAD) involved in FOXO4 binding and designed an optimized peptide inhibitor (CPP-CAND) with improved cell permeability. CPP-CAND showed high selectivity and potency in inducing apoptosis in senescent cells by disrupting FOXO4-p53 foci and activating caspase pathways. It is effective against senescent cancer cells induced by doxorubicin and cisplatin, highlighting its potential as a senolytic drug. Thus, CPP-CAND is a promising therapeutic candidate with improved selectivity, efficacy, and cost-effectiveness.
{"title":"Peptide inhibitors targeting FOXO4-p53 interactions and inducing senescent cancer cell-specific apoptosis","authors":"Donghoon Kang, Yeji Lim, Dabin Ahn, Jaeseok Lee, Chin-Ju Park","doi":"10.1101/2024.09.04.610228","DOIUrl":"https://doi.org/10.1101/2024.09.04.610228","url":null,"abstract":"Cellular senescence, marked by irreversible cell cycle arrest and the secretion of proinflammatory factors, contributes to aging and cancer recurrence. Chemotherapy can also induce senescence; senescent cells often resist apoptosis and promote tumor recurrence. The interaction between FOXO4 and p53 sustains cell survival. Herein, we used biophysical techniques and conducted cellular experiments to develop a peptide inhibitor targeting this interaction to eliminate senescent cancer cells. We identified key regions in the p53 transactivation domain (TAD) involved in FOXO4 binding and designed an optimized peptide inhibitor (CPP-CAND) with improved cell permeability. CPP-CAND showed high selectivity and potency in inducing apoptosis in senescent cells by disrupting FOXO4-p53 foci and activating caspase pathways. It is effective against senescent cancer cells induced by doxorubicin and cisplatin, highlighting its potential as a senolytic drug. Thus, CPP-CAND is a promising therapeutic candidate with improved selectivity, efficacy, and cost-effectiveness.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176100","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 : 2024-09-04DOI: 10.1101/2024.09.04.611303
Sagnik Sen, Amar Thaker, Dewight Williams, Po-Lin Chiu, Brent L. Nannenga
Visualizing the structure of the protein-inorganic interface is critically important for our more complete understanding of biomineralization. Unfortunately, there are limited approaches for the direct and detailed study of biomolecules that interact with inorganic materials. Here we use single particle cryo-EM to study the protein-nanoparticle interactions of human light chain ferritin and visualize the high-resolution details of the protein-inorganic interface. In this work, we determined the 2.85 Å structure of human light chain ferritin bound to its native iron oxide nanoparticle substrate. The resulting cryo-EM maps confirmed and enhanced previously proposed interactions of the protein with the material along the B-helix, and revealed new interaction at the C-terminus of light chain ferritin. This work sheds new light on the mechanisms of ferritin biomineralization and further demonstrates the application of cryo-EM for the study of protein-inorganic systems.
可视化蛋白质-无机界面结构对于我们更全面地了解生物矿化至关重要。遗憾的是，直接详细研究与无机材料相互作用的生物大分子的方法非常有限。在这里，我们利用单颗粒低温电子显微镜研究了人类轻链铁蛋白的蛋白质-纳米颗粒相互作用，并对蛋白质-无机界面的高分辨率细节进行了可视化。在这项工作中，我们确定了人类轻链铁蛋白与其原生氧化铁纳米颗粒基质结合的 2.85 Å 结构。所绘制的低温电子显微镜图证实并增强了之前提出的蛋白质与材料沿 B 螺旋的相互作用，并揭示了轻链铁蛋白 C 端新的相互作用。这项研究揭示了铁蛋白生物矿化的新机制，并进一步证明了低温电子显微镜在蛋白质-无机系统研究中的应用。
{"title":"Observation of the protein-inorganic interface of ferritin by cryo-electron microscopy","authors":"Sagnik Sen, Amar Thaker, Dewight Williams, Po-Lin Chiu, Brent L. Nannenga","doi":"10.1101/2024.09.04.611303","DOIUrl":"https://doi.org/10.1101/2024.09.04.611303","url":null,"abstract":"Visualizing the structure of the protein-inorganic interface is critically important for our more complete understanding of biomineralization. Unfortunately, there are limited approaches for the direct and detailed study of biomolecules that interact with inorganic materials. Here we use single particle cryo-EM to study the protein-nanoparticle interactions of human light chain ferritin and visualize the high-resolution details of the protein-inorganic interface. In this work, we determined the 2.85 Å structure of human light chain ferritin bound to its native iron oxide nanoparticle substrate. The resulting cryo-EM maps confirmed and enhanced previously proposed interactions of the protein with the material along the B-helix, and revealed new interaction at the C-terminus of light chain ferritin. This work sheds new light on the mechanisms of ferritin biomineralization and further demonstrates the application of cryo-EM for the study of protein-inorganic systems.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176099","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 : 2024-09-04DOI: 10.1101/2024.09.04.611296
Vincent Nicholson, Kenny H Nguyen, Edith Gollub, Mary McCoy, Feng Yu, Alex S Holehouse, Shahar Sukenik, Thomas C Boothby
Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.
{"title":"LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation","authors":"Vincent Nicholson, Kenny H Nguyen, Edith Gollub, Mary McCoy, Feng Yu, Alex S Holehouse, Shahar Sukenik, Thomas C Boothby","doi":"10.1101/2024.09.04.611296","DOIUrl":"https://doi.org/10.1101/2024.09.04.611296","url":null,"abstract":"Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176096","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 : 2024-09-04DOI: 10.1101/2024.09.04.611280
Manxi Yang, Mushfeqa Iqfath, Frederick Nguele Meke, Zihan Qu, Emerson L. Hernly, Pei Su, Zhong-Yin Zhang, Julia Laskin
Mass spectrometry imaging (MSI) is a powerful technique for label-free spatial mapping of multiple classes of biomolecules in tissue sections. However, differences in desorption and ionization efficiency of different classes of molecules make it challenging to simultaneously map biomolecules at each omics layer in the same tissue sample. Herein, we present a correlative imaging method using nanospray desorption electrospray ionization (nano-DESI) MSI, which enables the spatial mapping of lipids, metabolites, peptides, and proteins with cellular-level spatial resolution in a single tissue section. We demonstrate the molecular profiling of specific cell types and identify truncated peptides in mouse pancreatic tissue. Distinct chemical gradients of peptides and lipids extending from endocrine cells to exocrine cells indicate their different roles in endocrine-exocrine crosstalk and intracellular signaling. The results underscore the power of the developed imaging approach for spatial multi-omics analysis that provides deep insights into cellular diversity and the intricate molecular interactions that occur within heterogenous biological tissues.
{"title":"Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues","authors":"Manxi Yang, Mushfeqa Iqfath, Frederick Nguele Meke, Zihan Qu, Emerson L. Hernly, Pei Su, Zhong-Yin Zhang, Julia Laskin","doi":"10.1101/2024.09.04.611280","DOIUrl":"https://doi.org/10.1101/2024.09.04.611280","url":null,"abstract":"Mass spectrometry imaging (MSI) is a powerful technique for label-free spatial mapping of multiple classes of biomolecules in tissue sections. However, differences in desorption and ionization efficiency of different classes of molecules make it challenging to simultaneously map biomolecules at each omics layer in the same tissue sample. Herein, we present a correlative imaging method using nanospray desorption electrospray ionization (nano-DESI) MSI, which enables the spatial mapping of lipids, metabolites, peptides, and proteins with cellular-level spatial resolution in a single tissue section. We demonstrate the molecular profiling of specific cell types and identify truncated peptides in mouse pancreatic tissue. Distinct chemical gradients of peptides and lipids extending from endocrine cells to exocrine cells indicate their different roles in endocrine-exocrine crosstalk and intracellular signaling. The results underscore the power of the developed imaging approach for spatial multi-omics analysis that provides deep insights into cellular diversity and the intricate molecular interactions that occur within heterogenous biological tissues.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176095","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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