Pub Date : 2024-03-07DOI: 10.1021/acschembio.3c00781
Yushu Gu, Miaomiao Liu, Linlin Ma and Ronald J. Quinn*,
The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.
{"title":"Advancing Kir4.2 Channel Ligand Identification through Collision-Induced Affinity Selection Mass Spectrometry","authors":"Yushu Gu, Miaomiao Liu, Linlin Ma and Ronald J. Quinn*, ","doi":"10.1021/acschembio.3c00781","DOIUrl":"10.1021/acschembio.3c00781","url":null,"abstract":"<p >The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00781","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140048139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-05DOI: 10.1021/acschembio.3c00649
Angy Liseth Dávalos, José David Rivera Echeverri, Denize C. Favaro, Ronaldo Junio de Oliveira, Gustavo Penteado Battesini Carretero, Caroline Lacerda, Iolanda Midea Cuccovia, Marcus Vinicius Cangussu Cardoso, Chuck S. Farah and Roberto Kopke Salinas*,
The understanding of protein–protein interaction mechanisms is key to the atomistic description of cell signaling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Nt behaves as a random coil, while VirB9Ct is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence, and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Nt coupled folding and binding mechanism. Qualitative data analysis suggested that VirB9Ct preferentially binds to VirB7Nt by way of a conformational selection mechanism at lower temperatures. However, at higher temperatures, energy barriers between different VirB9Ct conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may provide alternative pathways toward the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.
了解蛋白质与蛋白质之间的相互作用机制是从原子角度描述细胞信号传导途径和开发新药的关键。在这方面,内在无序蛋白在结合时折叠的机制引起了人们的关注。VirB9 C 端结构域(VirB9Ct)和 VirB7 N 端结构域(VirB7Nt)与 VirB10 结合形成 IV 型分泌系统注射体的外膜核心复合物。尽管形成了稳定而坚硬的复合物,但 VirB7Nt 表现为随机线圈,而 VirB9Ct 在自由状态下具有内在的动态性。在这里,我们结合核磁共振、停流荧光和计算机模拟,使用基于结构的模型来描述 VirB9Ct-VirB7Nt 的耦合折叠和结合机制。定性数据分析表明,在较低温度下,VirB9Ct 通过构象选择机制优先与 VirB7Nt 结合。然而,在较高温度下,不同 VirB9Ct 构象之间的能量障碍更容易被超越。在这些条件下,非原生初始相遇复合物的形成可能提供了通向原生复合物构象的替代途径。这些观察结果突显了折叠与结合之间的密切关系,使人们注意到两个分子伙伴必须在崎岖不平的构象能谱上寻找最有利的分子内和分子间相互作用,沿着这个能谱,多个中间产物可能会导致最终的原生状态。
{"title":"Uncovering the Association Mechanism between Two Intrinsically Flexible Proteins","authors":"Angy Liseth Dávalos, José David Rivera Echeverri, Denize C. Favaro, Ronaldo Junio de Oliveira, Gustavo Penteado Battesini Carretero, Caroline Lacerda, Iolanda Midea Cuccovia, Marcus Vinicius Cangussu Cardoso, Chuck S. Farah and Roberto Kopke Salinas*, ","doi":"10.1021/acschembio.3c00649","DOIUrl":"10.1021/acschembio.3c00649","url":null,"abstract":"<p >The understanding of protein–protein interaction mechanisms is key to the atomistic description of cell signaling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9<sup>Ct</sup>) and the VirB7 N-terminal motif (VirB7<sup>Nt</sup>) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7<sup>Nt</sup> behaves as a random coil, while VirB9<sup>Ct</sup> is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence, and computer simulations using structure-based models to characterize the VirB9<sup>Ct</sup>-VirB7<sup>Nt</sup> coupled folding and binding mechanism. Qualitative data analysis suggested that VirB9<sup>Ct</sup> preferentially binds to VirB7<sup>Nt</sup> by way of a conformational selection mechanism at lower temperatures. However, at higher temperatures, energy barriers between different VirB9<sup>Ct</sup> conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may provide alternative pathways toward the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140074969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-05DOI: 10.1021/acschembio.3c00724
Lindsay E. Guzmán, C. J. Cambier, Tan-Yun Cheng, Kubra F. Naqvi, Michael U. Shiloh, D. Branch Moody and Carolyn R. Bertozzi*,
Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host–pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.
{"title":"Bioorthogonal Metabolic Labeling of the Virulence Factor Phenolic Glycolipid in Mycobacteria","authors":"Lindsay E. Guzmán, C. J. Cambier, Tan-Yun Cheng, Kubra F. Naqvi, Michael U. Shiloh, D. Branch Moody and Carolyn R. Bertozzi*, ","doi":"10.1021/acschembio.3c00724","DOIUrl":"10.1021/acschembio.3c00724","url":null,"abstract":"<p >Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical <i>Mycobacterium tuberculosis</i> strains. PGL is also found in <i>Mycobacterium marinum</i>, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host–pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor <i>p</i>-hydroxybenzoic acid (<i>p</i>HB) with an azide group (3-azido <i>p</i>HB). When fed to mycobacteria, 3-azido <i>p</i>HB was incorporated into the cell surface, which could then be visualized <i>via</i> the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido <i>p</i>HB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing <i>M. marinum</i> and <i>M. tuberculosis</i> strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00724","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140038152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-28DOI: 10.1021/acschembio.3c00795
Angelica Faith L. Suarez, Thi Quynh Ngoc Nguyen, Litao Chang, Yi Wei Tooh, Rubin How Sheng Yong, Li Chuan Leow, Ivan Yu Fan Koh, Huiyi Chen, Jeffery Wei Heng Koh, Arunachalam Selvanayagam, Vernon Lim, Yi En Tan, Irene Agatha, Fernaldo R. Winnerdy and Brandon I. Morinaka*,
Enzymes catalyzing peptide macrocyclization are important biochemical tools in drug discovery. The three-residue cyclophane-forming enzymes (3-CyFEs) are an emerging family of post-translational modifying enzymes that catalyze the formation of three-residue peptide cyclophanes. In this report, we introduce three additional 3-CyFEs, including ChlB, WnsB, and FnnB, that catalyze cyclophane formation on Tyr, Trp, and Phe, respectively. To understand the promiscuity of these enzymes and those previously reported (MscB, HaaB, and YxdB), we tested single amino acid substitutions at the three-residue motif of modification (Ω1X2X3, Ω1 = aromatic). Collectively, we observe that substrate promiscuity is observed at the Ω1 and X2 positions, but a greater specificity is observed for the X3 residue. Two nonnative cyclophane products were characterized showing a Phe-C3 to Arg-Cβ and His-C2 to Pro-Cβ cross-links, respectively. We also tested the leader dependence of selected 3-CyFEs and show that a predicted helix region is important for cyclophane formation. These results demonstrate the biocatalytic potential of these maturases and allow rational design of substrates to obtain a diverse array of genetically encoded 3-residue cyclophanes.
{"title":"Functional and Promiscuity Studies of Three-Residue Cyclophane Forming Enzymes Show Nonnative C–C Cross-Linked Products and Leader-Dependent Cyclization","authors":"Angelica Faith L. Suarez, Thi Quynh Ngoc Nguyen, Litao Chang, Yi Wei Tooh, Rubin How Sheng Yong, Li Chuan Leow, Ivan Yu Fan Koh, Huiyi Chen, Jeffery Wei Heng Koh, Arunachalam Selvanayagam, Vernon Lim, Yi En Tan, Irene Agatha, Fernaldo R. Winnerdy and Brandon I. Morinaka*, ","doi":"10.1021/acschembio.3c00795","DOIUrl":"10.1021/acschembio.3c00795","url":null,"abstract":"<p >Enzymes catalyzing peptide macrocyclization are important biochemical tools in drug discovery. The <u>three</u>-residue <u>cy</u>clophane-<u>f</u>orming <u>e</u>nzyme<u>s</u> (3-CyFEs) are an emerging family of post-translational modifying enzymes that catalyze the formation of three-residue peptide cyclophanes. In this report, we introduce three additional 3-CyFEs, including ChlB, WnsB, and FnnB, that catalyze cyclophane formation on Tyr, Trp, and Phe, respectively. To understand the promiscuity of these enzymes and those previously reported (MscB, HaaB, and YxdB), we tested single amino acid substitutions at the three-residue motif of modification (Ω1X2X3, Ω1 = aromatic). Collectively, we observe that substrate promiscuity is observed at the Ω1 and X2 positions, but a greater specificity is observed for the X3 residue. Two nonnative cyclophane products were characterized showing a Phe-C3 to Arg-Cβ and His-C2 to Pro-Cβ cross-links, respectively. We also tested the leader dependence of selected 3-CyFEs and show that a predicted helix region is important for cyclophane formation. These results demonstrate the biocatalytic potential of these maturases and allow rational design of substrates to obtain a diverse array of genetically encoded 3-residue cyclophanes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-28DOI: 10.1021/acschembio.3c00775
Yuqi Chen, Angela Simeone, Larry Melidis, Sergio Martinez Cuesta, David Tannahill and Shankar Balasubramanian*,
Four-stranded G-quadruplexes (G4s) are DNA secondary structures that can form in the human genome. G4 structures have been detected in gene promoters and are associated with transcriptionally active chromatin and the recruitment of transcription factors and chromatin remodelers. We adopted a controlled, synthetic biology approach to understand how G4s can influence transcription. We stably integrated G4-forming sequences into the promoter of a synthetic reporter gene and inserted these into the genome of human cells. The integrated G4 sequences were shown to fold into a G4 structure within a cellular genomic context. We demonstrate that G4 structure formation within a gene promoter stimulates transcription compared to the corresponding G4-negative control promoter in a way that is not dependent on primary sequence or inherent G-richness. Systematic variation in the stability of folded G4s showed that in this system, transcriptional levels increased with higher stability of the G4 structure. By creating and manipulating a chromosomally integrated synthetic promoter, we have shown that G4 structure formation in a defined gene promoter can cause gene transcription to increase, which aligns with earlier observational correlations reported in the literature linking G4s to active transcription.
{"title":"An Upstream G-Quadruplex DNA Structure Can Stimulate Gene Transcription","authors":"Yuqi Chen, Angela Simeone, Larry Melidis, Sergio Martinez Cuesta, David Tannahill and Shankar Balasubramanian*, ","doi":"10.1021/acschembio.3c00775","DOIUrl":"10.1021/acschembio.3c00775","url":null,"abstract":"<p >Four-stranded G-quadruplexes (G4s) are DNA secondary structures that can form in the human genome. G4 structures have been detected in gene promoters and are associated with transcriptionally active chromatin and the recruitment of transcription factors and chromatin remodelers. We adopted a controlled, synthetic biology approach to understand how G4s can influence transcription. We stably integrated G4-forming sequences into the promoter of a synthetic reporter gene and inserted these into the genome of human cells. The integrated G4 sequences were shown to fold into a G4 structure within a cellular genomic context. We demonstrate that G4 structure formation within a gene promoter stimulates transcription compared to the corresponding G4-negative control promoter in a way that is not dependent on primary sequence or inherent G-richness. Systematic variation in the stability of folded G4s showed that in this system, transcriptional levels increased with higher stability of the G4 structure. By creating and manipulating a chromosomally integrated synthetic promoter, we have shown that G4 structure formation in a defined gene promoter can cause gene transcription to increase, which aligns with earlier observational correlations reported in the literature linking G4s to active transcription.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00775","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139988620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-27DOI: 10.1021/acschembio.3c00435
Julius Blechar, Vanessa de Jesus, Boris Fürtig, Martin Hengesbach* and Harald Schwalbe*,
Translational riboswitches located in the 5′ UTR of the messenger RNA (mRNA) regulate translation through variation of the accessibility of the ribosome binding site (RBS). These are the result of conformational changes in the riboswitch RNA governed by ligand binding. Here, we use a combination of single-molecule colocalization techniques (Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) and Single-Molecule Kinetic Analysis of Ribosome Binding (SiM-KARB)) and microscale thermophoresis (MST) to investigate the adenine-sensing riboswitch in Vibrio vulnificus, focusing on the changes of accessibility between the ligand-free and ligand-bound states. We show that both methods faithfully report on the accessibility of the RBS within the riboswitch and that both methods identify an increase in accessibility upon adenine binding. Expanding on the regulatory context, we show the impact of the ribosomal protein S1 on the unwinding of the RNA secondary structure, thereby favoring ribosome binding even for the apo state. The determined rate constants suggest that binding of the ribosome is faster than the time required to change from the ON state to the OFF state, a prerequisite for efficient regulation decision.
{"title":"Shine–Dalgarno Accessibility Governs Ribosome Binding to the Adenine Riboswitch","authors":"Julius Blechar, Vanessa de Jesus, Boris Fürtig, Martin Hengesbach* and Harald Schwalbe*, ","doi":"10.1021/acschembio.3c00435","DOIUrl":"10.1021/acschembio.3c00435","url":null,"abstract":"<p >Translational riboswitches located in the 5′ UTR of the messenger RNA (mRNA) regulate translation through variation of the accessibility of the ribosome binding site (RBS). These are the result of conformational changes in the riboswitch RNA governed by ligand binding. Here, we use a combination of single-molecule colocalization techniques (Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) and Single-Molecule Kinetic Analysis of Ribosome Binding (SiM-KARB)) and microscale thermophoresis (MST) to investigate the adenine-sensing riboswitch in <i>Vibrio vulnificus</i>, focusing on the changes of accessibility between the ligand-free and ligand-bound states. We show that both methods faithfully report on the accessibility of the RBS within the riboswitch and that both methods identify an increase in accessibility upon adenine binding. Expanding on the regulatory context, we show the impact of the ribosomal protein S1 on the unwinding of the RNA secondary structure, thereby favoring ribosome binding even for the apo state. The determined rate constants suggest that binding of the ribosome is faster than the time required to change from the ON state to the OFF state, a prerequisite for efficient regulation decision.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139981971","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-27DOI: 10.1021/acschembio.3c00779
Miao Yu, T.M. Simon Tang, Lila Ghamsari, Graham Yuen, Claudio Scuoppo, Jim A. Rotolo, Barry J. Kappel and Jody M. Mason*,
Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C–C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.
{"title":"Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor","authors":"Miao Yu, T.M. Simon Tang, Lila Ghamsari, Graham Yuen, Claudio Scuoppo, Jim A. Rotolo, Barry J. Kappel and Jody M. Mason*, ","doi":"10.1021/acschembio.3c00779","DOIUrl":"10.1021/acschembio.3c00779","url":null,"abstract":"<p >Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C–C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five <i>a</i> positions to identify those that provided improved binding, with all <i>e/g</i> positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten <i>e/g</i> positions with 3 options. Similarly, during <i>e/g</i> library screening, <i>a</i> positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto <i>e/g</i> options provided. The combined <i>a/e/g</i> library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (<i>T</i><sub>m</sub> = 60 °C; <i>K</i><sub>d</sub> = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined <i>a/e/g</i> exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00779","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139981970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-26DOI: 10.1021/acschembio.3c00676
Saddam Y. Khatik, Sarupa Roy and Seergazhi G. Srivatsan*,
Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called “one-label, one-technique” approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2′-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.
{"title":"Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA","authors":"Saddam Y. Khatik, Sarupa Roy and Seergazhi G. Srivatsan*, ","doi":"10.1021/acschembio.3c00676","DOIUrl":"10.1021/acschembio.3c00676","url":null,"abstract":"<p >Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called “one-label, one-technique” approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and <sup>19</sup>F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2′-deoxyuridine, which serves as an excellent fluorescent and <sup>19</sup>F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and <sup>19</sup>F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and <sup>19</sup>F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139970249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nonribosomal peptide synthetases (NRPSs) are sophisticated molecular machines that biosynthesize peptide drugs. In attempts to generate new bioactive compounds, some parts of NRPSs have been successfully manipulated, but especially the influence of condensation (C-)domains on substrate specificity remains enigmatic and poorly controlled. To understand the influence of C-domains on substrate preference, we extensively evaluated the peptide formation of C-domain mutants in a bimodular NRPS system. Thus, we identified three key mutations that govern the preference for stereoconfiguration and side-chain identity. These mutations show similar effects in three different C-domains (GrsB1, TycB1, and SrfAC) when di- or pentapeptides are synthesized in vitro or in vivo. Strikingly, mutation E386L allows the stereopreference to be switched from d- to l-configured donor substrates. Our findings provide valuable insights into how cryptic specificity filters in C-domains can be re-engineered to clear roadblocks for NRPS engineering and enable the production of novel bioactive compounds.
{"title":"Controlling Substrate- and Stereospecificity of Condensation Domains in Nonribosomal Peptide Synthetases","authors":"Huiyun Peng, Julian Schmiederer, Xiuqiang Chen, Gianni Panagiotou and Hajo Kries*, ","doi":"10.1021/acschembio.3c00678","DOIUrl":"10.1021/acschembio.3c00678","url":null,"abstract":"<p >Nonribosomal peptide synthetases (NRPSs) are sophisticated molecular machines that biosynthesize peptide drugs. In attempts to generate new bioactive compounds, some parts of NRPSs have been successfully manipulated, but especially the influence of condensation (C-)domains on substrate specificity remains enigmatic and poorly controlled. To understand the influence of C-domains on substrate preference, we extensively evaluated the peptide formation of C-domain mutants in a bimodular NRPS system. Thus, we identified three key mutations that govern the preference for stereoconfiguration and side-chain identity. These mutations show similar effects in three different C-domains (GrsB1, TycB1, and SrfAC) when di- or pentapeptides are synthesized <i>in vitro</i> or <i>in vivo</i>. Strikingly, mutation E386L allows the stereopreference to be switched from <span>d</span>- to <span>l</span>-configured donor substrates. Our findings provide valuable insights into how cryptic specificity filters in C-domains can be re-engineered to clear roadblocks for NRPS engineering and enable the production of novel bioactive compounds.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139939098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-23DOI: 10.1021/acschembio.3c00542
Fabiola V. De León González, Marie E. Boddington, Joshua M. Kofsky, Martha I. Prindl and Chantelle J. Capicciotti*,
Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1F383A. The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.
{"title":"Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs","authors":"Fabiola V. De León González, Marie E. Boddington, Joshua M. Kofsky, Martha I. Prindl and Chantelle J. Capicciotti*, ","doi":"10.1021/acschembio.3c00542","DOIUrl":"10.1021/acschembio.3c00542","url":null,"abstract":"<p >Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1<sup>F383A</sup>. The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139939099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}