Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612398
Alejandro J. Brenes
The Coefficient of Variation (CV) is a measure that is frequently used to assess data dispersion for mass spectrometry-based proteomics. In the current era of burgeoning technical developments, there has been an increased focus on using CVs to measure the quantitative accuracy of the new methods. Thus, it has also become important to define a set of guidelines on how to calculate and report the CVs.This perspective shows the effects that the CV equation, as well as software parameters can have on data dispersion and CVs, highlighting the importance of reporting all these variables within the methods section. It also proposes a set of recommendations to calculate and report CVs for technical studies where the main objective is to benchmark technical developments with a focus on precision. To assist in this process a novel R package to calculate CVs ( proteomicsCV ) is also included.
{"title":"Calculating and Reporting Coefficients of Variation for DIA-based Proteomics","authors":"Alejandro J. Brenes","doi":"10.1101/2024.09.11.612398","DOIUrl":"https://doi.org/10.1101/2024.09.11.612398","url":null,"abstract":"The Coefficient of Variation (CV) is a measure that is frequently used to assess data dispersion for mass spectrometry-based proteomics. In the current era of burgeoning technical developments, there has been an increased focus on using CVs to measure the quantitative accuracy of the new methods. Thus, it has also become important to define a set of guidelines on how to calculate and report the CVs.This perspective shows the effects that the CV equation, as well as software parameters can have on data dispersion and CVs, highlighting the importance of reporting all these variables within the methods section. It also proposes a set of recommendations to calculate and report CVs for technical studies where the main objective is to benchmark technical developments with a focus on precision. To assist in this process a novel R package to calculate CVs ( proteomicsCV ) is also included.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254901","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 : 2024-09-13DOI: 10.1101/2024.09.11.612554
Vinay K Menon, Joy Wu, Alex Alonzo, Kaitlyn Rogers, Kevin L. Scrudders, Suriya Selvarajan, Andrew Walke, Rajasree Kundu, Ankona Datta, Shalini Therese Low-Nam
Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low abundance membrane constituents with different arrangements of phosphate groups around an inositol head group that regulate a large number of signaling pathways. Many strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interaction with binding partners. We implement a peptide-based ratiometric sensor for the detection of PI(4,5)P2 lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P2 densities down to physiological levels. The sensor is membrane permeable and easily applicable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P2 levels and distributions in a mutation-specific manner. The rapid implementation of this quantitative sensing strategy to cellular studies of cellular signaling, membrane organization and dynamics should be broadly applicable.
{"title":"Direct measurement of PIP2 densities in biological membranes using a peptide-based sensor","authors":"Vinay K Menon, Joy Wu, Alex Alonzo, Kaitlyn Rogers, Kevin L. Scrudders, Suriya Selvarajan, Andrew Walke, Rajasree Kundu, Ankona Datta, Shalini Therese Low-Nam","doi":"10.1101/2024.09.11.612554","DOIUrl":"https://doi.org/10.1101/2024.09.11.612554","url":null,"abstract":"Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low abundance membrane constituents with different arrangements of phosphate groups around an inositol head group that regulate a large number of signaling pathways. Many strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interaction with binding partners. We implement a peptide-based ratiometric sensor for the detection of PI(4,5)P2 lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P2 densities down to physiological levels. The sensor is membrane permeable and easily applicable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P2 levels and distributions in a mutation-specific manner. The rapid implementation of this quantitative sensing strategy to cellular studies of cellular signaling, membrane organization and dynamics should be broadly applicable.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254929","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 : 2024-09-13DOI: 10.1101/2024.09.13.612689
Sarah R Stevenson, Svetomir B. Tzokov, Indrajit Lahiri, Kathryn R Ayscough, Per A Bullough
The core component of the actin cytoskeleton is the globular protein G-actin, which reversibly polymerises into filaments (F-actin). Budding yeast possesses a single actin which shares 87-89% sequence identity with vertebrate actin isoforms. Previous structural studies indicate very close overlap of main-chain backbones. Intriguingly however, substitution of yeast ACT1 with vertebrate β-cytoplasmic actin severely disrupts cell function and substitution with a skeletal muscle isoform is lethal. Here we report a 2.5 Angstrom structure of budding yeast F-actin. Previously, unresolved side-chain information now highlights four main differences in the comparison of yeast and vertebrate ADP F-actins: a more open nucleotide binding pocket; a more solvent exposed C-terminus; a rearrangement of intersubunit binding interactions in the vicinity of the D-loop and changes in the hydrogen bonding network in the vicinity of histidine 73 (yeast actin) and methyl-histidine 73 (vertebrate actin).
{"title":"CryoEM Reconstruction of Yeast ADP-Actin Filament at 2.5 Angstrom resolution. A comparison with mammalian and avian F-actin.","authors":"Sarah R Stevenson, Svetomir B. Tzokov, Indrajit Lahiri, Kathryn R Ayscough, Per A Bullough","doi":"10.1101/2024.09.13.612689","DOIUrl":"https://doi.org/10.1101/2024.09.13.612689","url":null,"abstract":"The core component of the actin cytoskeleton is the globular protein G-actin, which reversibly polymerises into filaments (F-actin). Budding yeast possesses a single actin which shares 87-89% sequence identity with vertebrate actin isoforms. Previous structural studies indicate very close overlap of main-chain backbones. Intriguingly however, substitution of yeast ACT1 with vertebrate β-cytoplasmic actin severely disrupts cell function and substitution with a skeletal muscle isoform is lethal. Here we report a 2.5 Angstrom structure of budding yeast F-actin. Previously, unresolved side-chain information now highlights four main differences in the comparison of yeast and vertebrate ADP F-actins: a more open nucleotide binding pocket; a more solvent exposed C-terminus; a rearrangement of intersubunit binding interactions in the vicinity of the D-loop and changes in the hydrogen bonding network in the vicinity of histidine 73 (yeast actin) and methyl-histidine 73 (vertebrate actin).","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254926","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 : 2024-09-13DOI: 10.1101/2024.09.13.612773
Matthias Gloegl, Aditya Krishnakumar, Robert Ragotte, Inna Goreshnik, Brian Coventry, Asim K Bera, Alex Kang, Emily Joyce, Green Ahn, Buwei Huang, Wei Yang, Wei Chen, Mariana Garcia Sanchez, Brian Koepnick, David Baker
Despite progress in the design of protein binding proteins, the shape matching of binder to target has not yet reached that of highly evolved native protein-protein complexes, and previous design efforts have failed for hard targets such as the TNF receptor (TNFR1) that have relatively flat and polar surfaces. We reasoned that free diffusion starting from random noise could enable generation of extensive shape-matching binders to challenging targets, and tested this approach on TNFR1 and related super family members. The diffused TNFR1 binders have nanomolar affinities that increase to single-digit picomolar upon refinement by partial diffusion, and their specificities can be completely switched by partial diffusion in the context of other family members. The designs function as antagonists as monomers, and as superagonists when presented trivalently for OX40 and at higher valency for 4-1BB. The ability to design high -affinity and specific antagonists and agonists for a difficult but pharmacologically important class of proteins entirely in silico, without any large-scale screening or experimental optimization, presages a new era in which binders are made by computation rather than much more laborious and less controllable random screening approaches.
{"title":"Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists","authors":"Matthias Gloegl, Aditya Krishnakumar, Robert Ragotte, Inna Goreshnik, Brian Coventry, Asim K Bera, Alex Kang, Emily Joyce, Green Ahn, Buwei Huang, Wei Yang, Wei Chen, Mariana Garcia Sanchez, Brian Koepnick, David Baker","doi":"10.1101/2024.09.13.612773","DOIUrl":"https://doi.org/10.1101/2024.09.13.612773","url":null,"abstract":"Despite progress in the design of protein binding proteins, the shape matching of binder to target has not yet reached that of highly evolved native protein-protein complexes, and previous design efforts have failed for hard targets such as the TNF receptor (TNFR1) that have relatively flat and polar surfaces. We reasoned that free diffusion starting from random noise could enable generation of extensive shape-matching binders to challenging targets, and tested this approach on TNFR1 and related super family members. The diffused TNFR1 binders have nanomolar affinities that increase to single-digit picomolar upon refinement by partial diffusion, and their specificities can be completely switched by partial diffusion in the context of other family members. The designs function as antagonists as monomers, and as superagonists when presented trivalently for OX40 and at higher valency for 4-1BB. The ability to design high -affinity and specific antagonists and agonists for a difficult but pharmacologically important class of proteins entirely in silico, without any large-scale screening or experimental optimization, presages a new era in which binders are made by computation rather than much more laborious and less controllable random screening approaches.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142269048","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 : 2024-09-13DOI: 10.1101/2024.09.13.612799
Gizem Olcucu, Bastian Wollenhaupt, Dietrich Kohlheyer, Karl-Erich Jaeger, Ulrich Krauss
Efficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. In this contribution, we present a novel immobilization strategy utilizing in vivo produced, magnetic protein aggregates (MPAs). MPA production is facilitated by the expression of gene fusions consisting of genes encoding for the yellow fluorescent protein variant citrine and variants of the iron storage protein ferritin, including a magnetically enhanced ferritin mutant from Escherichia coli. Expression of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, which is driven by citrine-dependent dimerization of ferritin cages. Upon cell lysis, the assemblies coalesce in solution to form MPAs. The fusion of the mutant E. coli ferritin to citrine yields fluorescent, insoluble protein aggregates that display magnetic properties, verified by their attraction to neodymium magnets. We further demonstrate that these novel, fully in vivo produced protein aggregates can be magnetically purified without the need for ex vivo iron-loading. Utilizing a bait/prey strategy, MPAs were functionalized by the post-translational attachment of an alcohol dehydrogenase to the MPA particles to enable proof-of-concept for enzyme immobilization, giving rise to catalytically-active magnetic protein aggregates (CatMPAs). The resulting (Cat)MPAs could easily be obtained from crude cell extracts via centrifugation, or purified using magnetic columns, and exhibited superior stability. The strategy presented here therefore represents a highly modular method to produce magnetic enzyme immobilizates which can be obtained with high purity.
{"title":"Magnetic Protein Aggregates Generated by Supramolecular Assembly of Ferritin Cages - A Modular Strategy for the Immobilization of Enzymes","authors":"Gizem Olcucu, Bastian Wollenhaupt, Dietrich Kohlheyer, Karl-Erich Jaeger, Ulrich Krauss","doi":"10.1101/2024.09.13.612799","DOIUrl":"https://doi.org/10.1101/2024.09.13.612799","url":null,"abstract":"Efficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. In this contribution, we present a novel immobilization strategy utilizing in vivo produced, magnetic protein aggregates (MPAs). MPA production is facilitated by the expression of gene fusions consisting of genes encoding for the yellow fluorescent protein variant citrine and variants of the iron storage protein ferritin, including a magnetically enhanced ferritin mutant from Escherichia coli. Expression of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, which is driven by citrine-dependent dimerization of ferritin cages. Upon cell lysis, the assemblies coalesce in solution to form MPAs. The fusion of the mutant E. coli ferritin to citrine yields fluorescent, insoluble protein aggregates that display magnetic properties, verified by their attraction to neodymium magnets. We further demonstrate that these novel, fully in vivo produced protein aggregates can be magnetically purified without the need for ex vivo iron-loading. Utilizing a bait/prey strategy, MPAs were functionalized by the post-translational attachment of an alcohol dehydrogenase to the MPA particles to enable proof-of-concept for enzyme immobilization, giving rise to catalytically-active magnetic protein aggregates (CatMPAs). The resulting (Cat)MPAs could easily be obtained from crude cell extracts via centrifugation, or purified using magnetic columns, and exhibited superior stability. The strategy presented here therefore represents a highly modular method to produce magnetic enzyme immobilizates which can be obtained with high purity.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"201 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254928","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 : 2024-09-13DOI: 10.1101/2024.08.29.610163
Johnny Mendoza, Kazuhiro Yamada, Carmen Castillo, Catherine A. Wilhelm, Markos Koutmos
Cobalamin (vitamin B12) and its derivatives are used to power chemical transformations crucial for life. Among these essential reactivities are methylations, of which cobalamin-dependent methionine synthase (MS) is the canonical example. MS catalyzes three distinct methyl transfers central to one-carbon metabolism. Despite its importance in the biological methyl cycle and relevance to human health, fundamental studies on the molecular basis of MS catalysis have proven elusive due to substantial biochemical challenges associated with MS from traditional sources. Here, we leverage our previously established thermophilic model system (tMS), its remarkable stability, and its ability to bind non-native cobalamin cofactors to systematically capture previously unattainable conformations via crystallography, expanding the conformational ensemble of MS to include gating conformations and present the first structures of a cobalamin enzyme in action (folate demethylation and homocysteine methylation). We establish the role of the folate (Fol) domain and its associated linkers in triggering the structural transitions required for activity. Our work highlights the importance of linkers in mediating large-scale rearrangements that underpin the catalysis of improbable chemistries. By providing the first structural blueprints associated with two cobalamin-mediated methyl transfers, we lay the groundwork for exploring cobalamin's biocatalytic potential and provide a framework by which Nature harnesses dynamic motions to achieve dynamic chemical outcomes.
钴胺素（维生素 B12）及其衍生物被用于促进对生命至关重要的化学转化。在这些重要的化学反应中，甲基化是其中之一，依赖钴胺素的蛋氨酸合成酶（MS）就是典型的例子。MS 催化单碳代谢中三种不同的甲基转移。尽管 MS 在生物甲基循环中非常重要，而且与人类健康息息相关，但由于传统来源的 MS 在生化方面存在巨大挑战，因此有关 MS 催化的分子基础的基础研究一直难以开展。在这里，我们利用之前建立的嗜热模型系统（tMS）、其出色的稳定性及其结合非原生钴胺辅助因子的能力，通过晶体学系统地捕捉到了之前无法实现的构象，将 MS 的构象组合扩展到包括门控构象，并首次展示了钴胺酶在作用（叶酸去甲基化和同型半胱氨酸甲基化）中的结构。我们确定了叶酸（Fol）结构域及其相关连接体在触发活性所需的结构转换中的作用。我们的工作凸显了连接体在介导大规模重排方面的重要性，这些重排是催化不可能的化学反应的基础。通过首次提供与两种钴胺素介导的甲基转移相关的结构蓝图，我们为探索钴胺素的生物催化潜力奠定了基础，并提供了一个大自然利用动态运动实现动态化学结果的框架。
{"title":"Orchestrating Improbable Chemistries: Structural Snapshots of B12-Dependent Methionine Synthase's Catalytic Choreography","authors":"Johnny Mendoza, Kazuhiro Yamada, Carmen Castillo, Catherine A. Wilhelm, Markos Koutmos","doi":"10.1101/2024.08.29.610163","DOIUrl":"https://doi.org/10.1101/2024.08.29.610163","url":null,"abstract":"Cobalamin (vitamin B<sub>12</sub>) and its derivatives are used to power chemical transformations crucial for life. Among these essential reactivities are methylations, of which cobalamin-dependent methionine synthase (MS) is the canonical example. MS catalyzes three distinct methyl transfers central to one-carbon metabolism. Despite its importance in the biological methyl cycle and relevance to human health, fundamental studies on the molecular basis of MS catalysis have proven elusive due to substantial biochemical challenges associated with MS from traditional sources. Here, we leverage our previously established thermophilic model system (<em>t</em>MS), its remarkable stability, and its ability to bind non-native cobalamin cofactors to systematically capture previously unattainable conformations via crystallography, expanding the conformational ensemble of MS to include gating conformations and present the first structures of a cobalamin enzyme in action (folate demethylation and homocysteine methylation). We establish the role of the folate (Fol) domain and its associated linkers in triggering the structural transitions required for activity. Our work highlights the importance of linkers in mediating large-scale rearrangements that underpin the catalysis of improbable chemistries. By providing the first structural blueprints associated with two cobalamin-mediated methyl transfers, we lay the groundwork for exploring cobalamin's biocatalytic potential and provide a framework by which Nature harnesses dynamic motions to achieve dynamic chemical outcomes.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175885","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 : 2024-09-13DOI: 10.1101/2024.09.12.612761
Daniel J Pape, Kelly C Falls-Hubert, Ronald A Merrill, Adnan Ahmed, Qingwen Qian, Gavin R McGivney, Paulina Sobieralski, Adam J Rauckhorst, Ling Yang, Eric B Taylor
Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and 13C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.
{"title":"The mitochondrial dicarboxylate carrier mediates in vivo hepatic gluconeogenesis","authors":"Daniel J Pape, Kelly C Falls-Hubert, Ronald A Merrill, Adnan Ahmed, Qingwen Qian, Gavin R McGivney, Paulina Sobieralski, Adam J Rauckhorst, Ling Yang, Eric B Taylor","doi":"10.1101/2024.09.12.612761","DOIUrl":"https://doi.org/10.1101/2024.09.12.612761","url":null,"abstract":"Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and <sup>13</sup>C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254927","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 : 2024-09-12DOI: 10.1101/2024.09.12.612650
Pierre Raia, Kitaik Lee, Simon M Bartsch, Felix Rico-Resendiz, Dorothea Fiedler, Michael Hothorn
Inositol pyrophosphates are highly phosphorylated nutrient messengers. The final step of their biosynthesis is catalyzed by diphosphoinositol pentakisphosphate kinase (PPIP5K) enzymes, which are conserved among fungi, plants, and animals. PPIP5Ks contain an N-terminal kinase domain that generates the active messenger 1,5-InsP8 and a C-terminal phosphatase domain that participates in PP-InsP catabolism. The balance between kinase and phosphatase activities controls the cellular levels and signaling capacity of 1,5-InsP8. Here, we present crystal structures of the apo and substrate-bound Vip1 phosphatase domain from S. cerevisiae (ScVip1PD). ScVip1PD is a phytase-like inositol 1-pyrophosphate phosphatase with two conserved histidine phosphatase catalytic motifs. The enzyme has a strong preference for 1,5-InsP8 and is inhibited by inorganic phosphate. ScVip1PD has an alpha-helical insertion domain stabilized by a structural Zn2+ binding site, and a unique GAF domain that exists in an open and closed state, allowing channeling of the 1,5-InsP8 substrate to the active site. Mutations that alter the active site, that restrict the movement of the GAF domain or that modify the charge of the substrate channel significantly inhibit the activity of the yeast enzyme in vitro, and the function of the Arabidopsis PPIP5K VIH2 in planta. Structural analyses of full-length PPIP5Ks suggest that the kinase and phosphatase are independent enzymatic modules. Taken together, our work reveals the structure, enzymatic mechanism and regulation of eukaryotic PPIP5K phosphatases.
肌醇焦磷酸盐是高度磷酸化的营养信使。它们生物合成的最后一步是由二磷酸肌醇五磷酸激酶（PPIP5K）催化的，这种酶在真菌、植物和动物中都是保守的。PPIP5K 包含一个 N 端激酶结构域和一个 C 端磷酸酶结构域，前者产生活性信使 1,5-InsP8，后者参与 PP-InsP 的分解代谢。激酶和磷酸酶活性之间的平衡控制着 1,5-InsP8 的细胞水平和信号能力。在这里，我们展示了来自 S. cerevisiae（ScVip1PD）的 Vip1 磷酸酶结构域（apo）和与底物结合的 Vip1 磷酸酶结构域（substrate-bound Vip1 phosphatase domain）的晶体结构。ScVip1PD 是一种类似植物酶的肌醇 1-焦磷酸磷酸酶，具有两个保守的组氨酸磷酸酶催化基团。该酶对 1,5-InsP8具有强烈的偏好，并受到无机磷酸盐的抑制。ScVip1PD 有一个由结构性 Zn2+ 结合位点稳定的 alpha-helical 插入结构域，以及一个独特的 GAF 结构域，该结构域以开放和封闭状态存在，可将 1,5-InsP8 底物引导至活性位点。改变活性位点、限制 GAF 结构域移动或改变底物通道电荷的突变会显著抑制体外酵母酶的活性以及拟南芥 PPIP5K VIH2 在植物体内的功能。对全长 PPIP5K 的结构分析表明，激酶和磷酸酶是独立的酶模块。总之，我们的工作揭示了真核生物 PPIP5K 磷酸酶的结构、酶学机制和调控。
{"title":"A GAF domain mediates inositol pyrophosphate substrate channeling in PPIP5K phosphatases","authors":"Pierre Raia, Kitaik Lee, Simon M Bartsch, Felix Rico-Resendiz, Dorothea Fiedler, Michael Hothorn","doi":"10.1101/2024.09.12.612650","DOIUrl":"https://doi.org/10.1101/2024.09.12.612650","url":null,"abstract":"Inositol pyrophosphates are highly phosphorylated nutrient messengers. The final step of their biosynthesis is catalyzed by diphosphoinositol pentakisphosphate kinase (PPIP5K) enzymes, which are conserved among fungi, plants, and animals. PPIP5Ks contain an N-terminal kinase domain that generates the active messenger 1,5-InsP8 and a C-terminal phosphatase domain that participates in PP-InsP catabolism. The balance between kinase and phosphatase activities controls the cellular levels and signaling capacity of 1,5-InsP8. Here, we present crystal structures of the apo and substrate-bound Vip1 phosphatase domain from S. cerevisiae (ScVip1PD). ScVip1PD is a phytase-like inositol 1-pyrophosphate phosphatase with two conserved histidine phosphatase catalytic motifs. The enzyme has a strong preference for 1,5-InsP8 and is inhibited by inorganic phosphate. ScVip1PD has an alpha-helical insertion domain stabilized by a structural Zn2+ binding site, and a unique GAF domain that exists in an open and closed state, allowing channeling of the 1,5-InsP8 substrate to the active site. Mutations that alter the active site, that restrict the movement of the GAF domain or that modify the charge of the substrate channel significantly inhibit the activity of the yeast enzyme in vitro, and the function of the Arabidopsis PPIP5K VIH2 in planta. Structural analyses of full-length PPIP5Ks suggest that the kinase and phosphatase are independent enzymatic modules. Taken together, our work reveals the structure, enzymatic mechanism and regulation of eukaryotic PPIP5K phosphatases.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"40 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175886","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 : 2024-09-12DOI: 10.1101/2024.09.11.612515
Kenneth D Carr, Dane Evan D. Zambrano, Connor Weidle, Alex Goodson, Helen E Eisenach, Harley Pyles, Alexis Courbet, Neil P King, Andrew J Borst
Protein purification is essential in protein biochemistry, structural biology, and protein design. It enables the determination of protein structures, the study of biological mechanisms, and the biochemical and biophysical characterization of both natural and de novo designed proteins. Despite the broad application of various protein purification protocols, standard strategies can still encounter challenges, such as the unintended co-purification of unknown contaminants alongside the target protein. In particular, co-purification issues pose significant challenges for designed self-assembling protein nanomaterials, as it is difficult to determine whether unexpected observed geometries represent novel assembly states of the designed system, cross-contamination from other assemblies, or native proteins originating from the expression host. In this study, we assessed the ability of an automated structure-to-sequence pipeline to unambiguously identify an unknown co-purifying protein found across several purified designed protein samples. Using cryo-electron microscopy (Cryo-EM), ModelAngelo's sequence-agnostic automated model-building feature, and the Basic Local Alignment Search Tool (BLAST), we identified the unknown protein as dihydrolipoamide succinyltransferase (DLST). This identification was further confirmed by comparing the cryo-EM data with available DLST structures in the Protein Data Bank (PDB) and AlphaFold 3 predictions from the top BLAST hits. The clear identification of DLST informed our subsequent literature search and led to the rational modification of our protein purification protocol, ultimately enabling the exclusion of the contaminant from preparations of our target nanoparticle. This study demonstrates the successful application of a structure-to-sequence workflow, integrating Cryo-EM, ModelAngelo, protein BLAST, PDB structures, and AlphaFold 3 predictions, to identify and remove an unknown protein contaminant from multiple purified samples. It also highlights the broader potential of integrating Cryo-EM with AI-driven tools for accurate protein identification across various samples and contexts in protein science.
{"title":"Protein identification using cryo-EM and artificial intelligence guides improved sample purification","authors":"Kenneth D Carr, Dane Evan D. Zambrano, Connor Weidle, Alex Goodson, Helen E Eisenach, Harley Pyles, Alexis Courbet, Neil P King, Andrew J Borst","doi":"10.1101/2024.09.11.612515","DOIUrl":"https://doi.org/10.1101/2024.09.11.612515","url":null,"abstract":"Protein purification is essential in protein biochemistry, structural biology, and protein design. It enables the determination of protein structures, the study of biological mechanisms, and the biochemical and biophysical characterization of both natural and de novo designed proteins. Despite the broad application of various protein purification protocols, standard strategies can still encounter challenges, such as the unintended co-purification of unknown contaminants alongside the target protein. In particular, co-purification issues pose significant challenges for designed self-assembling protein nanomaterials, as it is difficult to determine whether unexpected observed geometries represent novel assembly states of the designed system, cross-contamination from other assemblies, or native proteins originating from the expression host. In this study, we assessed the ability of an automated structure-to-sequence pipeline to unambiguously identify an unknown co-purifying protein found across several purified designed protein samples. Using cryo-electron microscopy (Cryo-EM), ModelAngelo's sequence-agnostic automated model-building feature, and the Basic Local Alignment Search Tool (BLAST), we identified the unknown protein as dihydrolipoamide succinyltransferase (DLST). This identification was further confirmed by comparing the cryo-EM data with available DLST structures in the Protein Data Bank (PDB) and AlphaFold 3 predictions from the top BLAST hits. The clear identification of DLST informed our subsequent literature search and led to the rational modification of our protein purification protocol, ultimately enabling the exclusion of the contaminant from preparations of our target nanoparticle. This study demonstrates the successful application of a structure-to-sequence workflow, integrating Cryo-EM, ModelAngelo, protein BLAST, PDB structures, and AlphaFold 3 predictions, to identify and remove an unknown protein contaminant from multiple purified samples. It also highlights the broader potential of integrating Cryo-EM with AI-driven tools for accurate protein identification across various samples and contexts in protein science.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175889","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 : 2024-09-11DOI: 10.1101/2024.09.10.612210
Alain Boussac, Julien Selles, Miwa Sugiura
The active site for water oxidation in Photosystem II (PSII ) consists of a Mn4CaO5 cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states, from S0 to S4, in the water splitting process. O2 evolution occurs in the final S3TyrZ dot to S0TyrZ transition. Chloride is also involved in this mechanism. By using PSII from Thermosynechococcus elongatus in which both Ca and Cl have been substituted for Sr and Br, in order to slow down the S3TyrZdot to S0TyrZ + O2 transition, with a t1/2 ~ 5 ms at room temperature, it is shown that the kinetics of the recovery of a functional S0 has a tfrac12 also close to 5 ms. It is suggested that, similarly, the reformation of a functional S0 state follows the S3TyrZdot to S0TyrZ + O2 transition transition in CaCl-PSII and that the insertion of a new substrate molecule of water (O5) and protons does not require further delay.
{"title":"Kinetics of reformation of an S0 state capable of progressing to an S1 state after the O2 release by Photosystem II","authors":"Alain Boussac, Julien Selles, Miwa Sugiura","doi":"10.1101/2024.09.10.612210","DOIUrl":"https://doi.org/10.1101/2024.09.10.612210","url":null,"abstract":"The active site for water oxidation in Photosystem II (PSII ) consists of a Mn4CaO5 cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states, from S0 to S4, in the water splitting process. O2 evolution occurs in the final S3TyrZ dot to S0TyrZ transition. Chloride is also involved in this mechanism. By using PSII from Thermosynechococcus elongatus in which both Ca and Cl have been substituted for Sr and Br, in order to slow down the S3TyrZdot to S0TyrZ + O2 transition, with a t1/2 ~ 5 ms at room temperature, it is shown that the kinetics of the recovery of a functional S0 has a tfrac12 also close to 5 ms. It is suggested that, similarly, the reformation of a functional S0 state follows the S3TyrZdot to S0TyrZ + O2 transition transition in CaCl-PSII and that the insertion of a new substrate molecule of water (O5) and protons does not require further delay.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175904","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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