bioRxiv - Biochemistry最新文献_第8页

Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A 结核分枝杆菌必需转运体外排蛋白 A 的结构和抑制机制

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 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

{"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}

引用次数: 0

Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle 驱动蛋白-8 电机通过将其近端尾部结构域折叠成紧凑的螺旋束来实现二聚化

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 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

{"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}

引用次数: 0

Nsp1 stalls DNA Polymerase alpha at DNA hairpins Nsp1 在 DNA 发夹处阻滞 DNA 聚合酶 alpha

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.03.608162

Andrey G Baranovskiy, Lucia M Morstadt, Nigar D Babayeva, Tahir H. Tahirov

引用次数: 0

LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning LevSeq：快速生成用于定向进化和机器学习的序列功能数据

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 10.1101/2024.09.04.611255

Yueming Long, Ariane Mora, Emre Guersoy, Kadina E. Johnston, Francesca-Zhoufan Li, Frances H. Arnold

{"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}

引用次数: 0

Crystal structure and catalytic mechanism of PL35 family glycosaminoglycan lyases with an ultrabroad substrate spectrum 具有超宽底物谱的 PL35 家族糖胺聚糖裂解酶的晶体结构和催化机制

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 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

{"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}

引用次数: 0

Dynamics in the Phytophthora capsici effector AVR3a11 confirm the core WY domain fold 疫霉菌效应子 AVR3a11 的动态变化证实了核心 WY 结构域的折叠结构

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 10.1101/2024.09.04.611235

James Tolchard, Vicki S. Chambers, Laurence S. Boutemy, Mark J Banfield, Tharin M. A. Blumenschein

{"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}

引用次数: 0

Peptide inhibitors targeting FOXO4-p53 interactions and inducing senescent cancer cell-specific apoptosis 针对 FOXO4-p53 相互作用并诱导衰老癌细胞特异性凋亡的多肽抑制剂

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 10.1101/2024.09.04.610228

Donghoon Kang, Yeji Lim, Dabin Ahn, Jaeseok Lee, Chin-Ju Park

引用次数: 0

Observation of the protein-inorganic interface of ferritin by cryo-electron microscopy 用冷冻电镜观察铁蛋白的蛋白质无机界面

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 10.1101/2024.09.04.611303

Sagnik Sen, Amar Thaker, Dewight Williams, Po-Lin Chiu, Brent L. Nannenga

引用次数: 0

LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation LEA_4 motif单独或与协同共溶质共同发挥作用，在干燥过程中保护易变酶

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 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

{"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}

引用次数: 0

Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues 用于生物组织中单个细胞类型综合分子图谱的相关成像技术

bioRxiv - Biochemistry Pub Date : 2024-09-04 DOI: 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

{"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}

引用次数: 0