Biochemistry Biochemistry最新文献_第9页

Kinetic and Mechanistic Studies of Human Oligoadenylate Synthetase 1 人类寡腺苷酸合成酶 1 的动力学和机理研究

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-25 DOI: 10.1021/acs.biochem.4c0031110.1021/acs.biochem.4c00311

Ross L. Stein*,

{"title":"Kinetic and Mechanistic Studies of Human Oligoadenylate Synthetase 1","authors":"Ross L. Stein*, ","doi":"10.1021/acs.biochem.4c0031110.1021/acs.biochem.4c00311","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00311https://doi.org/10.1021/acs.biochem.4c00311","url":null,"abstract":"Oligoadenylate synthetase 1 (OAS1) catalyzes the dsRNA-dependent polymerization of ATP to form oligoadenylate, a second messenger of the innate immunity system. This paper reports kinetic and mechanistic studies of OAS1-catalyzed dimerization of ATP to form 2′-5′-diadenylate and pyrophosphate (PPi), the first step in ATP polymerization. Major findings include the following: (1) Reaction progress curves for the production of PPi are biphasic, characterized by a presteady-state lag followed by the linear, steady-state production of PPi. (2) The dependence of steady-state velocity on ATP concentration is sigmoidal and can be described by a rate law derived for a mechanism involving enzyme-catalyzed substrate dimerization. (3) Steady-state velocities were determined as a function of ATP concentration at fixed concentrations of poly(I:C), a synthetic dsRNA activator of OAS1. The data suggest a random mechanism in which either ATP or poly(I:C) can add first to the enzyme. (4) The dependence of klag on poly(I:C) and ATP concentration requires expansion of this mechanism to include slow conformational isomerization of various poly(I:C)- and ATP-bound complexes of inactive OAS1 to form complexes comprising an active enzyme, to ultimately form the reactive Michaelis complex of active OAS1, poly(I:C), and two molecules of ATP. Finally, within this complex, the two molecules of ATP dimerize to form 2′-5′-diadenylate and pyrophosphate. (5) The pH dependence and solvent deuterium isotope effect for kcat suggests that proton transfer occurs in the rate-limiting transition state, which likely involves proton abstraction from the 2′-hydroxyl of the adenylate acceptor ATP as the oxygen of this hydroxyl attacks the a-phosphate of the adenylate donor ATP in an SN2 fashion.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437308","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

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 : 2024-09-25 DOI: 10.1021/acs.biochem.4c0024710.1021/acs.biochem.4c00247

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

{"title":"Functional Validation of SAM Riboswitch Element A from Listeria monocytogenes","authors":"Ian Hall, Kaitlyn Zablock, Raeleen Sobetski, Chase A. Weidmann and Sarah C. Keane*, ","doi":"10.1021/acs.biochem.4c0024710.1021/acs.biochem.4c00247","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00247https://doi.org/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":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437309","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 : 2024-09-25 DOI: 10.1021/acs.biochem.4c0027110.1021/acs.biochem.4c00271

Lev Levintov, Biswajit Gorai and Harish Vashisth*,

{"title":"Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases","authors":"Lev Levintov, Biswajit Gorai and Harish Vashisth*, ","doi":"10.1021/acs.biochem.4c0027110.1021/acs.biochem.4c00271","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00271https://doi.org/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":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00271","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437310","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

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 (2S,3S)-4-氟戊氨酸均匀取代缬氨酸的大肠杆菌肽基脯氨酰异构酶 B 的 1.3 Å 晶体结构揭示了 CH2F 基团的结构守恒性和多重交错旋转体

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-24 DOI: 10.1021/acs.biochem.4c0034510.1021/acs.biochem.4c00345

Rebecca L. Frkic, Yi Jiun Tan, Ansis Maleckis, Nicholas F. Chilton, Gottfried Otting* and 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* and Colin J. Jackson*, ","doi":"10.1021/acs.biochem.4c0034510.1021/acs.biochem.4c00345","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00345https://doi.org/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":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437265","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

Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation Src家族激酶Hck与ATP位点抑制剂A-419259的共晶体化稳定了扩展激活环构象

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-24 DOI: 10.1021/acs.biochem.4c0032310.1021/acs.biochem.4c00323

Ari M. Selzer, John J. Alvarado and Thomas E. Smithgall*,

{"title":"Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation","authors":"Ari M. Selzer, John J. Alvarado and Thomas E. Smithgall*, ","doi":"10.1021/acs.biochem.4c0032310.1021/acs.biochem.4c00323","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00323https://doi.org/10.1021/acs.biochem.4c00323","url":null,"abstract":"Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å. This structure reveals the complete Hck active site in the presence of A-419259, including the αC-helix, the DFG motif, and the activation loop. A-419259 binds at the ATP-site of Hck and induces an overall closed conformation of the kinase with the regulatory SH3 and SH2 domains bound intramolecularly to their respective internal ligands. A-419259 stabilizes the DFG-in/αC-helix-out conformation observed previously with Hck and the pyrazolopyrimidine inhibitor PP1 (PDB: 1QCF). However, the activation loop conformations are distinct, with PP1 inducing a folded loop structure with the tyrosine autophosphorylation site (Tyr416) pointing into the ATP binding site, while A-419259 stabilizes an extended loop conformation with Tyr416 facing out into the solvent. Autophosphorylation also induces activation loop extension and significantly reduces the Hck sensitivity to PP1 but not A-419259. In cancer cells where Hck is constitutively active, the extended autophosphorylation loop may render Hck more sensitive to inhibitors like A-419259 which prefer this kinase conformation. More generally, these results provide additional insight into targeted kinase inhibitor design and how conformational preferences of inhibitors may impact selectivity and potency.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00323","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437421","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

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 : 2024-09-24 DOI: 10.1021/acs.biochem.4c0002210.1021/acs.biochem.4c00022

Joshua A. Broome, Nguyen P. Nguyen, Cassidy R. E. Baumung, Vincent C. Chen* and 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* and Eric A. C. Bushnell*, ","doi":"10.1021/acs.biochem.4c0002210.1021/acs.biochem.4c00022","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00022https://doi.org/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":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437420","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

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 : 2024-09-23 DOI: 10.1021/acs.biochem.4c0042710.1021/acs.biochem.4c00427

Bhagyesh Parmar, and , Dhiraj Bhatia*,

{"title":"Small Molecular Approaches for Cellular Reprogramming and Tissue Engineering: Functions as Mediators of the Cell Signaling Pathway","authors":"Bhagyesh Parmar,  and , Dhiraj Bhatia*, ","doi":"10.1021/acs.biochem.4c0042710.1021/acs.biochem.4c00427","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00427https://doi.org/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":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142437436","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

Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process 人类 CSTF2 RNA 识别动点域通过多步结合过程与 U 型 RNA 序列结合

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-21 DOI: 10.1021/acs.biochem.4c0040810.1021/acs.biochem.4c00408

Elahe Masoumzadeh, and , Michael P. Latham*,

{"title":"Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process","authors":"Elahe Masoumzadeh,  and , Michael P. Latham*, ","doi":"10.1021/acs.biochem.4c0040810.1021/acs.biochem.4c00408","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00408https://doi.org/10.1021/acs.biochem.4c00408","url":null,"abstract":"The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process. Given the importance of alternative cleavage and polyadenylation in increasing the diversity of mRNAs, the exact mechanism of binding of RNA to the RRM of CSTF2 remains unclear, particularly in the absence of a structure of this RRM bound to a native RNA substrate. Here, we performed a series of NMR titration and spin relaxation experiments, which were complemented by paramagnetic relaxation enhancement measurements and rigid-body docking, to characterize the interactions of the CSTF2 RRM with a U-rich ligand. Our results reveal a multistep binding process involving differences in ps-ns time scale dynamics and potential structural changes, particularly in the C-terminalα-helix. These results provide insights into how the CSTF2 RRM domain binds to U-rich RNA ligands and offer a greater understanding for the molecular basis of the regulation of pre-mRNA cleavage and polyadenylation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00408","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142407601","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

A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication 一种双位内体使 Vero 细胞不受蓖麻毒素侵袭

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-19 DOI: 10.1021/acs.biochem.4c0038510.1021/acs.biochem.4c00385

Timothy F. Czajka, David J. Vance, Renji Song and Nicholas J. Mantis*,

引用次数: 0

Collapsed State Mediates the Low Fidelity of the DNA Polymerase β I260 Mutant 塌缩状态介导了 DNA 聚合酶 β I260 突变体的低保真度

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-19 DOI: 10.1021/acs.biochem.4c0026310.1021/acs.biochem.4c00263

Carel Fijen*, Cristian Chavira, Khadijeh Alnajjar, Danielle L. Sawyer and Joann B. Sweasy*,

{"title":"Collapsed State Mediates the Low Fidelity of the DNA Polymerase β I260 Mutant","authors":"Carel Fijen*, Cristian Chavira, Khadijeh Alnajjar, Danielle L. Sawyer and Joann B. Sweasy*, ","doi":"10.1021/acs.biochem.4c0026310.1021/acs.biochem.4c00263","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00263https://doi.org/10.1021/acs.biochem.4c00263","url":null,"abstract":"DNA polymerase β (Pol β) fills single nucleotide gaps during base excision repair. Deficiencies in Pol β can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Förster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol β by creating a “collapsed” state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity and accurate DNA synthesis.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142402692","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