Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559–31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer–monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer–monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.
{"title":"Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution","authors":"Rainier-Numa Georges, Lionel Ballut, Nushin Aghajari, Laurence Hecquet, Franck Charmantray* and Bastien Doumèche*, ","doi":"10.1021/acs.biochem.3c00714","DOIUrl":"10.1021/acs.biochem.3c00714","url":null,"abstract":"<p >Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (<i>J Biol Chem</i>, <b>2010</b>, 285(41): 31559–31570) demonstrated that <i>Homo sapiens</i> TK (<i>hs</i>TK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with <i>hs</i>TK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of <i>Plasmodium falciparum</i>. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that <i>hs</i>TK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer–monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for <i>hs</i>TK containing only five residues in the crown. Residue mapping of the monomer–monomer interface and the active site reveals that <i>hs</i>TK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including <i>H. sapiens</i>) and <i>Schistosoma</i> sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141064374","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}
Pub Date : 2024-05-16DOI: 10.1021/acs.biochem.4c00080
Yi Jiun Tan, Elwy H. Abdelkader, Eliza Tarcoveanu, Ansis Maleckis, Christoph Nitsche and Gottfried Otting*,
Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl–prolyl cis–trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H–19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J.J. Am. Chem. Soc.1996, 118, 8700–8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5′-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.
{"title":"(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect","authors":"Yi Jiun Tan, Elwy H. Abdelkader, Eliza Tarcoveanu, Ansis Maleckis, Christoph Nitsche and Gottfried Otting*, ","doi":"10.1021/acs.biochem.4c00080","DOIUrl":"10.1021/acs.biochem.4c00080","url":null,"abstract":"<p >Global substitution of leucine for analogues containing CH<sub>2</sub>F instead of methyl groups delivers proteins with multiple sites for monitoring by <sup>19</sup>F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa <i>Escherichia coli</i> peptidyl–prolyl <i>cis–trans</i> isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2<i>S</i>,4<i>S</i>)-5-fluoroleucine, (2<i>S</i>,4<i>R</i>)-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. <sup>19</sup>F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The <sup>19</sup>F chemical shifts correlate with the three-bond <sup>1</sup>H–<sup>19</sup>F couplings (<sup>3</sup><i>J</i><sub>HF</sub>), providing the first experimental verification of the γ-gauche effect predicted by [<contrib-group><span>Feeney, J.</span></contrib-group> <cite><i>J. Am. Chem. Soc.</i></cite> <span>1996</span>, <em>118</em>, 8700–8706] and establishing the effect as the predominant determinant of the <sup>19</sup>F chemical shifts of CH<sub>2</sub>F groups. Individual CH<sub>2</sub>F groups can be confined to single rotameric states by the protein environment, but most CH<sub>2</sub>F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5′-difluoroleucine bias the CH<sub>2</sub>F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the <sup>19</sup>F chemical shift to the rotameric state of the CH<sub>2</sub>F groups potentially renders them particularly sensitive for detecting allosteric effects.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943153","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}
Pub Date : 2024-05-15DOI: 10.1021/acs.biochem.4c00157
Ting Jiang, Guanghua Wan, Haikun Zhang, Yadav Prasad Gyawali, Eric S. Underbakke and Changjian Feng*,
Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain–domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232–2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.
哺乳动物体内的一氧化氮合酶(NOS)是一个多结构域蛋白家族,其结构域间电子转移(IET)受结构域-结构域相互作用的控制。钙调蛋白(Calmodulin,CaM)与 NOS 的 FMN 和血红素结构域之间连接区的典型 CaM 结合位点结合,并允许系链 FMN 结构域运动,从而使输出状态下的 FMN-heme IET 在亚基间进行,以产生 NO。我们之前的交叉连接质谱(XL MS)研究结果表明,大鼠神经元 NOS(nNOS)还原酶构建体(一种单体蛋白)的 CaM 响应区存在特定位点的蛋白质动力学[Jiang 等,《生物化学》,2023 年,62 期,2232-2237]。在这项工作中,我们扩展了 XL MS 结构制图和 AlphaFold 结构预测的组合方法,以研究同源二聚体 nNOS 加氧酶/FMN(oxyFMN)构建体,这是 NOS 输出状态的一个既定模型。我们采用了基于平行反应监测(PRM)的定量 XL MS(qXL MS)来评估 CaM 诱导的结构域间动力学和相互作用的变化。通过将交联映射到顶部 AlphaFold 结构模型上,确定了亚基内交联,并通过比较交联二聚体和单体带中的相对丰度对其进行了补充。此外,无 CaM 和有 CaM 结合的 nNOS 样品对比显示,CaM 能促成亚基间 FMN-血红素对接复合物的形成,而且 CaM 结合能诱导整个 NOS 区域发生广泛的异构构象变化。此外,观察到的交联位点对离子强度的变化有特异性反应。这表明链间盐桥负责稳定和定向输出状态,以实现高效的 FMN-血红素 IET。综上所述,我们的靶向 qXL MS 结果揭示了 CaM 和离子强度调节 CaM/FMN/heme 复合物的特定动态变化,尤其是在亚基间域间 FMN-heme 相互作用的背景下。
{"title":"Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry","authors":"Ting Jiang, Guanghua Wan, Haikun Zhang, Yadav Prasad Gyawali, Eric S. Underbakke and Changjian Feng*, ","doi":"10.1021/acs.biochem.4c00157","DOIUrl":"10.1021/acs.biochem.4c00157","url":null,"abstract":"<p >Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain–domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., <i>Biochemistry</i>, 2023, 62, 2232–2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920442","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.4c00182
Tomoyasu Noji, Yoshihiro Chiba, Keisuke Saito and Hiroshi Ishikita*,
Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson–Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.
{"title":"Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin","authors":"Tomoyasu Noji, Yoshihiro Chiba, Keisuke Saito and Hiroshi Ishikita*, ","doi":"10.1021/acs.biochem.4c00182","DOIUrl":"10.1021/acs.biochem.4c00182","url":null,"abstract":"<p ><i>Exiguobacterium sibiricum</i> rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson–Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a p<i>K</i><sub>a</sub> value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H<sub>2</sub>O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920440","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.4c00161
Bundit Kamsri, Pharit Kamsri, Auradee Punkvang, Aunlika Chimprasit, Patchreenart Saparpakorn, Supa Hannongbua, James Spencer, A. Sofia F. Oliveira*, Adrian J. Mulholland* and Pornpan Pungpo*,
DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.
DNA 回旋酶催化 DNA 的负超螺旋,对细菌的 DNA 复制、转录和重组至关重要,是包括结核分枝杆菌在内的多种病原体的重要抗菌靶标。DNA 回旋酶是一种四聚体(A2B2)蛋白质,由两种亚基类型组成:回旋酶 A(GyrA)携带断裂重组活性位点,而回旋酶 B(GyrB)催化能量转移和 DNA 易位所需的 ATP 水解。GyrB ATP 酶结构域在 ATP 存在时会二聚化,以捕获转位的 DNA(T-DNA)片段,这是链传导的第一步,其中两个 ATP 之一的水解以及由此产生的无机磷酸的释放是限速过程。在这里,对结核杆菌 GyrB 的 43 kDa N 端二聚体片段进行的动态非平衡分子动力学(D-NEMD)模拟显示了 ATPase 位点的事件(结合核苷酸的解离/水解)是如何通过通信途径传播到 GyrB ATPase 结构域的其他重要功能区的。具体来说,我们的模拟确定了两条不同的途径,它们分别将 GyrB ATPase 位点与被认为在捕获 DNA 之前与 GyrA 相互作用的棒状杆菌特异性 C 环连接起来,以及与 GyrB 转导结构域的 C 端连接起来,后者又与负责与 GyrA 和中心结合的 G 段 DNA 相互作用的 C 端 GyrB 拓扑异构酶-primase(TOPRIM)结构域连接起来。ATPase 位点与二聚体 GyrB 的 C 环之间的联系与结核杆菌 DNA 回旋酶相对于其他细菌物种的不寻常特性是一致的。
{"title":"Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations","authors":"Bundit Kamsri, Pharit Kamsri, Auradee Punkvang, Aunlika Chimprasit, Patchreenart Saparpakorn, Supa Hannongbua, James Spencer, A. Sofia F. Oliveira*, Adrian J. Mulholland* and Pornpan Pungpo*, ","doi":"10.1021/acs.biochem.4c00161","DOIUrl":"10.1021/acs.biochem.4c00161","url":null,"abstract":"<p >DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including <i>Mycobacterium tuberculosis</i>, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A<sub>2</sub>B<sub>2</sub>) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of <i>M. tuberculosis</i> GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of <i>M. tuberculosis</i> DNA gyrase relative to those from other bacterial species.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140915301","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.4c00081
Rebecca L. Frkic, Yi Jiun Tan, Elwy H. Abdelkader, Ansis Maleckis, Eliza Tarcoveanu, Christoph Nitsche, Gottfried Otting* and Colin J. Jackson*,
Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis–trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5′-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
{"title":"Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in a Protein","authors":"Rebecca L. Frkic, Yi Jiun Tan, Elwy H. Abdelkader, Ansis Maleckis, Eliza Tarcoveanu, Christoph Nitsche, Gottfried Otting* and Colin J. Jackson*, ","doi":"10.1021/acs.biochem.4c00081","DOIUrl":"10.1021/acs.biochem.4c00081","url":null,"abstract":"<p >Proteins produced with leucine analogues, where CH<sub>2</sub>F groups substitute specific methyl groups, can readily be probed by <sup>19</sup>F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH<sub>2</sub>F groups is unclear. We present high-resolution crystal structures of <i>Escherichia coli</i> peptidyl-prolyl <i>cis</i>–<i>trans</i> isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2<i>S</i>,4<i>S</i>)-5-fluoroleucine, (2<i>S</i>,4<i>R</i>)-5-fluoroleucine, or 5,5′-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH<sub>2</sub>F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH<sub>3</sub> groups for CH<sub>2</sub>F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH<sub>2</sub>F groups can be halted at cryogenic temperatures.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920434","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.3c00643
Nibedita Ray Chaudhuri, and , Shubhra Ghosh Dastidar*,
Allostery is a fundamental mechanism driving biomolecular processes that holds significant therapeutic concern. Our study rigorously investigates how two distinct machine-learning algorithms uniquely classify two already close-to-active DFG-in states of TAK1, differing just by the presence or absence of its allosteric activator TAB1, from an ensemble mixture of conformations (obtained from 2.4 μs molecular dynamics (MD) simulations). The novelty, however, lies in understanding the deeper algorithmic potentials to systematically derive a diverse set of differential residue connectivity features that reconstruct the essential mechanistic architecture for TAK1-TAB1 allostery in such a close-to-active biochemical scenario. While the recursive, random forest-based workflow displays the potential of conducting discretized, hierarchical derivation of allosteric features, a multilayer perceptron-based approach gains considerable efficacy in revealing fluid connected patterns of features when hybridized with mutual information scoring. Interestingly, both pipelines benchmark similar directions of functional conformational changes for TAK1′s activation. The findings significantly advance the depth of mechanistic understanding by highlighting crucial activation signatures along a directed C-lobe → activation loop → ATP pocket channel of information flow, including (1) the αF-αE biterminal alignments and (2) the “catalytic” drift of the activation loop toward kinase active site. Besides, some novel allosteric hotspots (K253, Y206, N189, etc.) are further recognized as TAB1 sensors, transducers, and responders, including a benchmark E70 mutation site, precisely mapping the important structural segments for sequential allosteric execution. Hence, our work demonstrates how to navigate through greater structural depths and dimensions of dynamic allosteric machineries just by leveraging standard ML methods in suitable streamlined workflows adaptive to the specific system and objectives.
{"title":"Adaptive Workflows of Machine Learning Illuminate the Sequential Operation Mechanism of the TAK1′s Allosteric Network","authors":"Nibedita Ray Chaudhuri, and , Shubhra Ghosh Dastidar*, ","doi":"10.1021/acs.biochem.3c00643","DOIUrl":"10.1021/acs.biochem.3c00643","url":null,"abstract":"<p >Allostery is a fundamental mechanism driving biomolecular processes that holds significant therapeutic concern. Our study rigorously investigates how two distinct machine-learning algorithms uniquely classify two already close-to-active DFG-in states of TAK1, differing just by the presence or absence of its allosteric activator TAB1, from an ensemble mixture of conformations (obtained from 2.4 μs molecular dynamics (MD) simulations). The novelty, however, lies in understanding the deeper algorithmic potentials to systematically derive a diverse set of differential residue connectivity features that reconstruct the essential mechanistic architecture for TAK1-TAB1 allostery in such a close-to-active biochemical scenario. While the recursive, random forest-based workflow displays the potential of conducting discretized, hierarchical derivation of allosteric features, a multilayer perceptron-based approach gains considerable efficacy in revealing fluid connected patterns of features when hybridized with mutual information scoring. Interestingly, both pipelines benchmark similar directions of functional conformational changes for TAK1′s activation. The findings significantly advance the depth of mechanistic understanding by highlighting crucial activation signatures along a directed C-lobe → activation loop → ATP pocket channel of information flow, including (1) the αF-αE biterminal alignments and (2) the “catalytic” drift of the activation loop toward kinase active site. Besides, some novel allosteric hotspots (K253, Y206, N189, etc.) are further recognized as TAB1 sensors, transducers, and responders, including a benchmark E70 mutation site, precisely mapping the important structural segments for sequential allosteric execution. Hence, our work demonstrates how to navigate through greater structural depths and dimensions of dynamic allosteric machineries just by leveraging standard ML methods in suitable streamlined workflows adaptive to the specific system and objectives.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920318","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.4c00090
Dulmi Senanayaka, Danyun Zeng, Emre Deniz, Indunil K. Priyankara, Joceline Helmbreck, Owen Schneider, Aashay Mardikar, Aykut Uren and Nicholas J. Reiter*,
Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing’s sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug’s mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.
{"title":"Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates","authors":"Dulmi Senanayaka, Danyun Zeng, Emre Deniz, Indunil K. Priyankara, Joceline Helmbreck, Owen Schneider, Aashay Mardikar, Aykut Uren and Nicholas J. Reiter*, ","doi":"10.1021/acs.biochem.4c00090","DOIUrl":"10.1021/acs.biochem.4c00090","url":null,"abstract":"<p >Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing’s sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition <i>in vitro</i>, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (<i>K</i><sub>i</sub> = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug’s mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920399","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}
Pub Date : 2024-05-14DOI: 10.1021/acs.biochem.4c00042
Frederik Gouliaev, Nicolas Jonsson, Sarah Gersing, Michael Lisby, Kresten Lindorff-Larsen* and Rasmus Hartmann-Petersen*,
PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.
{"title":"Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant","authors":"Frederik Gouliaev, Nicolas Jonsson, Sarah Gersing, Michael Lisby, Kresten Lindorff-Larsen* and Rasmus Hartmann-Petersen*, ","doi":"10.1021/acs.biochem.4c00042","DOIUrl":"10.1021/acs.biochem.4c00042","url":null,"abstract":"<p ><i>PGM1</i>-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in <i>S. cerevisiae</i> and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140920437","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}
Pub Date : 2024-05-10DOI: 10.1021/acs.biochem.3c00716
Jake M. Peterson, Scott T. Becker, Collin A. O’Leary, Puneet Juneja, Yang Yang and Walter N. Moss*,
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed −1 ribosomal frameshifting (−1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to −1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site–a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.
严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)的移帧刺激元件(FSE)是程序化-1 核糖体移帧(-1 PRF)和优化病毒效力所必需的。FSE 有大量依赖于上下文的交替构象,但其中对-1 PRF 最关键的两个结构是一个衰减发夹和一个三茎 H 型假结结构。仅假结的晶体结构就以螺旋堆叠的线性结构中的三个 RNA 茎为特征,而包括上游七聚体滑动位点在内的 6.9 Å Cryo-EM 结构则导致两个茎之间的弯曲。我们之前的研究暗示了一个扩展的上游多分支环,其中包括衰减器发夹和滑动位点--这是一种之前未建模的构象。我们的目的是通过计算和中分辨率冷冻电镜方法,提供一个 6.1 Å 的冷冻电镜结构,以线性假结结构和动态上游多分支环路为特征,进一步说明 SARS-CoV-2 FSE 的来龙去脉。
{"title":"Structure of the SARS-CoV-2 Frameshift Stimulatory Element with an Upstream Multibranch Loop","authors":"Jake M. Peterson, Scott T. Becker, Collin A. O’Leary, Puneet Juneja, Yang Yang and Walter N. Moss*, ","doi":"10.1021/acs.biochem.3c00716","DOIUrl":"10.1021/acs.biochem.3c00716","url":null,"abstract":"<p >The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed −1 ribosomal frameshifting (−1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to −1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site–a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140896673","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}