Pub Date : 2024-06-25DOI: 10.1021/acs.biochem.4c00194
Xi Wang, Luis Marcelo F Holthauzen, Jonathan M Paz-Villatoro, Karina G Bien, Binhan Yu, Junji Iwahara
The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.
HMGB1 蛋白通常作为 DNA 合子协助 DNA 修复酶和转录因子,但在某些细胞刺激下可从细胞核转移到细胞质甚至细胞外空间。引发 HMGB1 转位的因素之一是其在核定位序列附近被蛋白激酶 C(PKC)磷酸化,但 HMGB1 的确切修饰位点仍不明确。在本研究中,我们利用光谱学方法研究了 HMGB1 磷酸化及其对 HMGB1 蛋白分子特性的影响。我们对全长 HMGB1 蛋白的核磁共振(NMR)数据显示,PKC 对 HMGB1 的 DNA 结合结构域之一的 A-box 结构域进行了特异性磷酸化。S46 和 S53 的磷酸化尤其有效。在较长的反应时间内,PKC 磷酸化了 HMGB1 A-box 结构域内的其他一些残基。我们的荧光结合试验表明,磷酸化显著降低了 HMGB1 与 DNA 的结合亲和力。根据 HMGB1-DNA 复合物的晶体结构,这种效应可归因于 S46 侧链上带负电荷的磷酸基团与 DNA 主干之间的静电排斥作用。我们的数据还显示,磷酸化破坏了 A-box 结构域的折叠稳定性。因此,PKC 磷酸化会削弱 HMGB1 的 DNA 结合亲和力和折叠稳定性。
{"title":"Phosphorylation by Protein Kinase C Weakens DNA-Binding Affinity and Folding Stability of the HMGB1 Protein.","authors":"Xi Wang, Luis Marcelo F Holthauzen, Jonathan M Paz-Villatoro, Karina G Bien, Binhan Yu, Junji Iwahara","doi":"10.1021/acs.biochem.4c00194","DOIUrl":"10.1021/acs.biochem.4c00194","url":null,"abstract":"<p><p>The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448989","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 : 2024-06-24DOI: 10.1021/acs.biochem.4c00168
Marcelo D Polêto, Kylie D Allen, Justin A Lemkul
Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.
{"title":"Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F<sub>430</sub> Modifications.","authors":"Marcelo D Polêto, Kylie D Allen, Justin A Lemkul","doi":"10.1021/acs.biochem.4c00168","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00168","url":null,"abstract":"<p><p>Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F<sub>430</sub>, a nickel-containing tetrahydrocorphin. Several modified versions of F<sub>430</sub> have been discovered, including the 17<sup>2</sup>-methylthio-F<sub>430</sub> (mtF<sub>430</sub>) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from <i>Methanosarcina acetivorans</i> and ANME-1 when bound to the canonical F<sub>430</sub> compared to 17<sup>2</sup>-thioether coenzyme F<sub>430</sub> variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 17<sup>2</sup> methylthio modification in ANME-1 MCR. Modifications at the 17<sup>2</sup> position disrupt the canonical substrate positioning in <i>M. acetivorans</i> MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F<sub>430</sub> variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH<sub>3</sub>-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141445536","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 : 2024-06-22DOI: 10.1021/acs.biochem.4c00149
Mackenzie C. R. Denton, Natasha P. Murphy, Brenna Norton-Baker, Mauro Lua, Harrison Steel and Gregg T. Beckham*,
Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1–2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.
{"title":"Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis","authors":"Mackenzie C. R. Denton, Natasha P. Murphy, Brenna Norton-Baker, Mauro Lua, Harrison Steel and Gregg T. Beckham*, ","doi":"10.1021/acs.biochem.4c00149","DOIUrl":"10.1021/acs.biochem.4c00149","url":null,"abstract":"<p >Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1–2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00149","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141440035","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 : 2024-06-19DOI: 10.1021/acs.biochem.4c00166
Juan Pan, Eliott S. Wenger, Chi-Yun Lin, Bo Zhang, Debangsu Sil, Irene Schaperdoth, Setareh Saryazdi, Robert B. Grossman, Carsten Krebs* and J. Martin Bollinger Jr.*,
N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C–H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7–H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7–H cleavage and oxacyclization.
{"title":"An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO","authors":"Juan Pan, Eliott S. Wenger, Chi-Yun Lin, Bo Zhang, Debangsu Sil, Irene Schaperdoth, Setareh Saryazdi, Robert B. Grossman, Carsten Krebs* and J. Martin Bollinger Jr.*, ","doi":"10.1021/acs.biochem.4c00166","DOIUrl":"10.1021/acs.biochem.4c00166","url":null,"abstract":"<p ><i>N</i>-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-<i>exo</i>-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C–H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H<b>·</b> abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H<b>·</b> from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the <i>second</i> ferryl complex is not enhanced in <sup>2</sup>H<sub>2</sub>O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7–H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7–H cleavage and oxacyclization.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425666","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 : 2024-06-18DOI: 10.1021/acs.biochem.4c00066
Jessica L. Taylor, Pedro H. Ayres-Galhardo and Breann L. Brown*,
The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5′-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.
在某些细菌和真核生物中,氨基乙酰丙酸合成酶(ALAS)通过催化甘氨酸和琥珀酰-CoA缩合生成氨基乙酰丙酸,从而启动血红素的生物合成。在人类,负责在红细胞发育过程中产生血红素的 ALAS 同工酶是红细胞特异性 ALAS2 同工酶。由于 ALAS2 在红细胞生成过程中的重要作用,人类 ALAS2(hALAS2)功能的变化可导致两种不同的血液疾病。X连锁性红细胞性贫血是由ALAS2功能缺失引起的,而X连锁性原卟啉症则是由ALAS2功能获得引起的。有趣的是,ALAS2 C端延伸部分的突变可能与这两种疾病有关。在这里,我们研究了之前报道的两种 C 端功能缺失变体(hALAS2 V562A 和 M567I)介导的酶功能障碍的分子基础。我们发现,这些变异不会导致严重的结构紊乱,但 V562A 的酶稳定性会降低。此外,我们还发现这两个变体在加入 5'-磷酸吡哆醛(PLP)辅助因子后,酶的稳定性会适度增加。与野生型 hALAS2 相比,这些变体与 PLP 和单个底物的结合存在差异。虽然 hALAS2 V562A 在体外是一种更活跃的酶,但它与琥珀酰-CoA 结合的效率较低。相比之下,M567I 突变显著改变了底物结合的合作性。结合之前报道的基于细胞的研究,我们的工作揭示了 hALAS2 C 端突变对正常血红素生物合成所需的 ALA 生成产生负面影响的分子基础。
{"title":"Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease","authors":"Jessica L. Taylor, Pedro H. Ayres-Galhardo and Breann L. Brown*, ","doi":"10.1021/acs.biochem.4c00066","DOIUrl":"10.1021/acs.biochem.4c00066","url":null,"abstract":"<p >The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5′-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme <i>in vitro</i>, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416539","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}
Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer–graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer–GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin–GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.
人血清白蛋白(HSA)是一种蛋白质载体,可运输多种药物和营养物质。糖化 HSA(GHSA)的含量被用作糖尿病的生物标志物。要量化 GHSA 的含量,基于荧光石墨烯的灵敏传感器是一种成功的方法。在灵敏传感器中,关键机制是白蛋白从灵敏配体-石墨烯复合物中的吸附/解吸。最近,有报道称石墨烯量子点(GQD)可作为一种适配体吸附剂。由于石墨烯量子点的尺寸与吸附剂相当,因此有足够的吸引力来探索将石墨烯量子点作为白蛋白吸附传感器一部分的可能性。因此,我们在此进行了分子动力学(MD)模拟,以揭示白蛋白与一种吸附剂-GQD 复合物的分子结合机制。研究了白蛋白选择性适配体(GQDA)饱和的 GQD,并对 GHSA 和 HSA 进行了比较研究,以了解糖化的影响。观察到白蛋白与 GQDA 快速、自发地结合。虽然在两种白蛋白上都没有发现特定的 GQDA 结合位点,但用于结合的残基仅限于 HSA 的结构域 I 和 III 以及 GHSA 的结构域 II 和 III。研究发现,白蛋白更倾向于与适配体而不是 GQD 结合。赖氨酸和精氨酸是造成结合的主要原因。我们还发现 GLC 从所有 GHSA 轨迹中解离,这突显了 GQDA 在干扰 Sudlow 位点 I 的配体结合亲和力方面的作用。本研究获得的启示将有助于未来糖尿病相应传感器的设计。
{"title":"Aggregation of Apo/Glycated Human Serum Albumins and Aptamer-Saturated Graphene Quantum Dot: A Simulation Study","authors":"Sirin Sittiwanichai, Chanya Archapraditkul, Deanpen Japrung, Yasuteru Shigeta, Toshifumi Mori* and Prapasiri Pongprayoon*, ","doi":"10.1021/acs.biochem.4c00155","DOIUrl":"10.1021/acs.biochem.4c00155","url":null,"abstract":"<p >Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer–graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer–GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin–GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416538","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 : 2024-06-17DOI: 10.1021/acs.biochem.4c00165
Jake W. Saunders, Adam M. Damry, Vanessa Vongsouthi, Matthew A. Spence, Rebecca L. Frkic, Chloe Gomez, Patrick A. Yates, Dana S. Matthews, Nobuhiko Tokuriki, Malcolm D. McLeod and Colin J. Jackson*,
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
{"title":"Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design","authors":"Jake W. Saunders, Adam M. Damry, Vanessa Vongsouthi, Matthew A. Spence, Rebecca L. Frkic, Chloe Gomez, Patrick A. Yates, Dana S. Matthews, Nobuhiko Tokuriki, Malcolm D. McLeod and Colin J. Jackson*, ","doi":"10.1021/acs.biochem.4c00165","DOIUrl":"10.1021/acs.biochem.4c00165","url":null,"abstract":"<p >The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from <i>Ideonella sakaiensis</i> carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416540","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 : 2024-06-17DOI: 10.1021/acs.biochem.4c00177
Joshua R. Miller, Elizabeth C. Schnorrenberg, Cole Aschenbrener, Brian G. Fox and Thomas C. Brunold*,
In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys–Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys–Tyr cross-link during repeated enzyme turnover increases CDO’s catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys–Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.
{"title":"Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157","authors":"Joshua R. Miller, Elizabeth C. Schnorrenberg, Cole Aschenbrener, Brian G. Fox and Thomas C. Brunold*, ","doi":"10.1021/acs.biochem.4c00177","DOIUrl":"10.1021/acs.biochem.4c00177","url":null,"abstract":"<p >In mammals, <span>l</span>-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (<i>Mus musculus</i> CDO numbering) and a nearby tyrosine at position 157 (Cys–Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys–Tyr cross-link during repeated enzyme turnover increases CDO’s catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys–Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O<sub>2</sub>, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416541","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 : 2024-06-13DOI: 10.1021/acs.biochem.3c00728
Elijah D. Humphrey, and , Maxim V. Sukhodolets*,
In growing E. coli cells, the transcription–translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed “fast” procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP’s α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n “head-to-tail” polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells’ active growth.
在生长中的大肠杆菌细胞中,转录-翻译复合物(TTC)会形成特征性的病灶;然而,这些超结构的确切分子组成尚不确定。在此,我们报告说,在我们最近开发的大肠杆菌 RNA 聚合酶(RP)"快速 "纯化程序中,一部分 RP 的 α/RpoA 亚基脱离了核心 RP 复合物,并与携带核酸结合蛋白 Hfq 和核糖体蛋白 S6 的多蛋白超结构共聚。我们的研究表明,这些大型多蛋白集合体的主要成分是固定蛋白拷贝数(Hfq6)n≥8 的复合物;这些复合物的结构高度一致,与之前描述的(Hfq6)n "头对尾 "聚合物截然不同。我们描述了如何纯化这些结构均匀的新型 (Hfq6)n≥8 复合物,使其接近均一,并证明它们还含有小的非蛋白分子和附属 S6。我们证明 Hfq、S6 和 RP 具有相似的溶解度曲线,并提出证据表明 Hfq C 端在超结构形成中的作用。总之,我们的数据为了解细菌细胞活跃生长过程中可能作为转录复合物和核糖体支架的大分子组装体的组成提供了新的见解。
{"title":"Isolation and Partial Characterization of Novel, Structurally Uniform (Hfq6)n≥8 Assemblies Carrying Accessory Transcription and Translation Factors","authors":"Elijah D. Humphrey, and , Maxim V. Sukhodolets*, ","doi":"10.1021/acs.biochem.3c00728","DOIUrl":"10.1021/acs.biochem.3c00728","url":null,"abstract":"<p >In growing <i>E. coli</i> cells, the transcription–translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed “fast” procedures for purification of <i>E. coli</i> RNA polymerase (RP), a fraction of the RP’s α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq<sub>6</sub>)<sub>n≥8</sub> complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq<sub>6</sub>)<sub>n</sub> “head-to-tail” polymers. We describe purification of these novel, structurally uniform (Hfq<sub>6</sub>)<sub>n≥8</sub> complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells’ active growth.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141309488","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 : 2024-06-12DOI: 10.1021/acs.biochem.3c00665
Elsa D. M. Hien, Adrien Chauvier, Patrick St-Pierre and Daniel A. Lafontaine*,
Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.
{"title":"Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli","authors":"Elsa D. M. Hien, Adrien Chauvier, Patrick St-Pierre and Daniel A. Lafontaine*, ","doi":"10.1021/acs.biochem.3c00665","DOIUrl":"10.1021/acs.biochem.3c00665","url":null,"abstract":"<p >Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing <i>thiC</i> riboswitch in <i>Escherichia coli</i> by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the <i>thiC</i> riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141304775","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}