Biochemistry Biochemistry最新文献_第6页

Functional Validation of SAM Riboswitch Element A from Listeria monocytogenes. 单核细胞增生李斯特菌 SAM 核糖开关元件 A 的功能验证。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-25 DOI: 10.1021/acs.biochem.4c00247

Ian Hall, Kaitlyn Zablock, Raeleen Sobetski, Chase A Weidmann, Sarah C Keane

{"title":"Functional Validation of SAM Riboswitch Element A from Listeria monocytogenes.","authors":"Ian Hall, Kaitlyn Zablock, Raeleen Sobetski, Chase A Weidmann, Sarah C Keane","doi":"10.1021/acs.biochem.4c00247","DOIUrl":"10.1021/acs.biochem.4c00247","url":null,"abstract":"SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may \"dampen\" the expression of genes for methionine import but likely does not turn them \"OFF\".","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337288","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Gaining Insight into the Catalytic Mechanism of the R132H IDH1 Mutant: A Synergistic DFT Cluster and Experimental Investigation. 深入了解 R132H IDH1 突变体的催化机理：DFT 簇和实验研究的协同作用。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-24 DOI: 10.1021/acs.biochem.4c00022

Joshua A Broome, Nguyen P Nguyen, Cassidy R E Baumung, Vincent C Chen, Eric A C Bushnell

{"title":"Gaining Insight into the Catalytic Mechanism of the R132H IDH1 Mutant: A Synergistic DFT Cluster and Experimental Investigation.","authors":"Joshua A Broome, Nguyen P Nguyen, Cassidy R E Baumung, Vincent C Chen, Eric A C Bushnell","doi":"10.1021/acs.biochem.4c00022","DOIUrl":"10.1021/acs.biochem.4c00022","url":null,"abstract":"Human isocitrate dehydrogenase 1 (IDH1) is an enzyme that is found in humans that plays a critical role in aerobic metabolism. As a part of the citric acid cycle, IDH1 becomes responsible for catalyzing the oxidative decarboxylation of isocitrate to form α-ketoglutarate (αKG), with nicotinamide adenine dinucleotide phosphate (NADP+) as a cofactor. Strikingly, mutations of the IDH1 enzyme have been discovered in several cancers including glioblastoma multiforme (GBM), a highly aggressive form of brain cancer. It has been experimentally determined that single-residue IDH1 mutations occur at a very high frequency in GBM. Specifically, the IDH1 R132H mutation is known to produce (D)2-hydroxyglutarate (2HG), a recognized oncometabolite. Using the previously determined catalytic mechanism of IDH1, a DFT QM model was developed to study the mechanistic properties of IDH1 R132H compared to wild type enzyme. Validating these insights, biochemical in vitro assays of metabolites produced by mutant vs wild type enzymes were measured and compared. From the results discussed herein, we discuss the mechanistic impact of mutations in IDH1 on its ability to catalyze the formation of αKG and 2HG.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore 利用合成开启型红色荧光蛋白发色团选择性识别二聚体 NG16 平行 G 型四重结构

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 DOI: 10.1021/acs.biochem.4c0040710.1021/acs.biochem.4c00407

Nishant Kumar Choudhary, Shalini Gupta, Gourav Das, Avijit Sahoo, S. Harikrishna, Surajit Sinha and Kiran R. Gore*,

{"title":"Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore","authors":"Nishant Kumar Choudhary, Shalini Gupta, Gourav Das, Avijit Sahoo, S. Harikrishna, Surajit Sinha and Kiran R. Gore*, ","doi":"10.1021/acs.biochem.4c0040710.1021/acs.biochem.4c00407","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00407https://doi.org/10.1021/acs.biochem.4c00407","url":null,"abstract":"Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extraction of In-Cell β-Amyloid Fibrillar Aggregates for Studying Molecular-Level Structural Propagations Using Solid-State NMR Spectroscopy. 利用固态核磁共振波谱提取细胞内β-淀粉样蛋白纤维聚集体以研究分子级结构传播

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-30 DOI: 10.1021/acs.biochem.4c00395

June M Kenyaga, Wei Qiang

{"title":"Extraction of In-Cell β-Amyloid Fibrillar Aggregates for Studying Molecular-Level Structural Propagations Using Solid-State NMR Spectroscopy.","authors":"June M Kenyaga, Wei Qiang","doi":"10.1021/acs.biochem.4c00395","DOIUrl":"10.1021/acs.biochem.4c00395","url":null,"abstract":"Molecular-level structural polymorphisms of β-amyloid (Aβ) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aβ peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aβ concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aβ (Aβ40) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aβ40.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

1.3 Å Crystal Structure of E. coli Peptidyl-Prolyl Isomerase B with Uniform Substitution of Valine by (2S,3S)-4-Fluorovaline Reveals Structure Conservation and Multiple Staggered Rotamers of CH₂F Groups. 用 (2S,3S)-4-Fluorovaline 均匀取代缬氨酸的大肠杆菌肽脯氨酰异构酶 B 的 1.3 Å 晶体结构揭示了 CH2F 基团的结构守恒性和多个交错旋转体。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-24 DOI: 10.1021/acs.biochem.4c00345

Rebecca L Frkic, Yi Jiun Tan, Ansis Maleckis, Nicholas F Chilton, Gottfried Otting, Colin J Jackson

{"title":"1.3 Å Crystal Structure of E. coli Peptidyl-Prolyl Isomerase B with Uniform Substitution of Valine by (2S,3S)-4-Fluorovaline Reveals Structure Conservation and Multiple Staggered Rotamers of CH2F Groups.","authors":"Rebecca L Frkic, Yi Jiun Tan, Ansis Maleckis, Nicholas F Chilton, Gottfried Otting, Colin J Jackson","doi":"10.1021/acs.biochem.4c00345","DOIUrl":"10.1021/acs.biochem.4c00345","url":null,"abstract":"(2S,3S)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH3 group is substituted by a CH2F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a 13C-labeled CH2F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH2F groups invariably populate staggered rotamers. Most CH2F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. 19F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted 19F resonances correlate with large 3JHF coupling constants, confirming the impact of the γ-gauche effect on the signal dispersion. The present work is the second experimental verification of the effect and extends its validity to fluorovaline. The abundance of valine in proteins and structural conservation with FVal renders this valine analogue attractive for probing proteins by 19F NMR spectroscopy.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases. 受体酪氨酸激酶跨膜域的自发二聚化和不同包装模式。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-25 DOI: 10.1021/acs.biochem.4c00271

Lev Levintov, Biswajit Gorai, Harish Vashisth

{"title":"Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.","authors":"Lev Levintov, Biswajit Gorai, Harish Vashisth","doi":"10.1021/acs.biochem.4c00271","DOIUrl":"10.1021/acs.biochem.4c00271","url":null,"abstract":"The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483822/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mammalian Esterase Activity: Implications for Peptide Prodrugs. 哺乳动物的酯酶活性：肽原药的意义。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-10-03 DOI: 10.1021/acs.biochem.4c00446

Yana D Petri, Ruben Verresen, Clair S Gutierrez, Volga Kojasoy, Erika Zhang, Nile S Abularrage, Evans C Wralstad, Kaya R Weiser, Ronald T Raines

{"title":"Mammalian Esterase Activity: Implications for Peptide Prodrugs.","authors":"Yana D Petri, Ruben Verresen, Clair S Gutierrez, Volga Kojasoy, Erika Zhang, Nile S Abularrage, Evans C Wralstad, Kaya R Weiser, Ronald T Raines","doi":"10.1021/acs.biochem.4c00446","DOIUrl":"10.1021/acs.biochem.4c00446","url":null,"abstract":"As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11485170/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142363347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Small Molecular Approaches for Cellular Reprogramming and Tissue Engineering: Functions as Mediators of the Cell Signaling Pathway. 细胞重编程和组织工程的小分子方法：作为细胞信号通路媒介的功能。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-15 Epub Date: 2024-09-23 DOI: 10.1021/acs.biochem.4c00427

Bhagyesh Parmar, Dhiraj Bhatia

{"title":"Small Molecular Approaches for Cellular Reprogramming and Tissue Engineering: Functions as Mediators of the Cell Signaling Pathway.","authors":"Bhagyesh Parmar, Dhiraj Bhatia","doi":"10.1021/acs.biochem.4c00427","DOIUrl":"10.1021/acs.biochem.4c00427","url":null,"abstract":"Utilizing induced pluripotent stem cells (iPSCs) in drug screening and cell replacement therapy has emerged as a method with revolutionary applications. With the advent of patient-specific iPSCs and the subsequent development of cells that exhibit disease phenotypes, the focus of medication research will now shift toward the pathology of human diseases. Regular iPSCs can also be utilized to generate cells that assess the negative impacts of medications. These cells provide a much more precise and cost-efficient approach compared to many animal models. In this review, we explore the utilization of small-molecule drugs to enhance the growth of iPSCs and gain insights into the process of reprogramming. We mainly focus on the functions of small molecules in modulating different signaling pathways, thereby modulating cell fate. Understanding the way small molecule drugs interact with iPSC technology has the potential to significantly enhance the understanding of physiological pathways in stem cells and practical applications of iPSC-based therapy and screening systems, revolutionizing the treatment of diseases.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142306555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Unraveling the Bivalent and Rapid Interactions Between a Multivalent RNA Recognition Motif and RNA: A Kinetic Approach 揭示多价 RNA 识别基团与 RNA 之间的二价和快速相互作用：动力学方法

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-14 DOI: 10.1021/acs.biochem.4c0030110.1021/acs.biochem.4c00301

Guillermo Pérez-Ropero*, Anna Pérez-Ràfols, Tommasso Martelli, U. Helena Danielson and Jos Buijs,

{"title":"Unraveling the Bivalent and Rapid Interactions Between a Multivalent RNA Recognition Motif and RNA: A Kinetic Approach","authors":"Guillermo Pérez-Ropero*, Anna Pérez-Ràfols, Tommasso Martelli, U. Helena Danielson and Jos Buijs, ","doi":"10.1021/acs.biochem.4c0030110.1021/acs.biochem.4c00301","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00301https://doi.org/10.1021/acs.biochem.4c00301","url":null,"abstract":"The kinetics of the interaction between Musashi-1 (MSI1) and RNA have been characterized using surface plasmon resonance biosensor analysis. Truncated variants of human MSI1 encompassing the two homologous RNA recognition motifs (RRM1 and RRM2) in tandem (aa 1–200), and the two RRMs in isolation (aa 1–103 and aa 104–200, respectively) were produced. The proteins were injected over sensor surfaces with immobilized RNA, varying in sequence and length, and with one or two RRM binding motifs. The interactions of the individual RRMs with all RNA variants were well described by a 1:1 interaction model. The interaction between the MSI1 variant encompassing both RRM motifs was bivalent and rapid for all RNA variants. Due to difficulties in fitting this complex data using standard procedures, we devised a new method to quantify the interactions. It revealed that two RRMs in tandem resulted in a significantly longer residence time than a single RRM. It also showed that RNA with double UAG binding motifs and potential hairpin structures forms less stable bivalent complexes with MSI1 than the single UAG motif containing linear RNA. Substituting the UAG binding motif with a CAG sequence resulted in a reduction of the affinity of the individual RRMs, but for MSI1, this reduction was strongly enhanced, demonstrating the importance of bivalency for specificity. This study has provided new insights into the interaction between MSI1 and RNA and an understanding of how individual domains contribute to the overall interaction. It provides an explanation for why many RNA-binding proteins contain dual RRMs.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00301","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction 催化初始反马尔科夫尼科夫环化反应的倍半萜合成酶 Caryolan-1-ol Synthase 的晶体结构

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-14 DOI: 10.1021/acs.biochem.4c0054710.1021/acs.biochem.4c00547

Ramasamy P. Kumar, Jason O. Matos, Brandon Y. Black, William H. Ellenburg, Jiahua Chen, MacKenzie Patterson, Jacob A. Gehtman, Douglas L. Theobald*, Isaac J. Krauss* and Daniel D. Oprian*,

{"title":"Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction","authors":"Ramasamy P. Kumar, Jason O. Matos, Brandon Y. Black, William H. Ellenburg, Jiahua Chen, MacKenzie Patterson, Jacob A. Gehtman, Douglas L. Theobald*, Isaac J. Krauss* and Daniel D. Oprian*, ","doi":"10.1021/acs.biochem.4c0054710.1021/acs.biochem.4c00547","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00547https://doi.org/10.1021/acs.biochem.4c00547","url":null,"abstract":"In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1–C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1–C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0