Biochemistry Biochemistry最新文献

{"title":"Insight into the Mechanism of d-Glucose Accelerated Exchange in GLUT1 from Molecular Dynamics Simulations.","authors":"Carmen Domene, Brian Wiley, Saul Gonzalez-Resines, Richard J Naftalin","doi":"10.1021/acs.biochem.4c00502","DOIUrl":"10.1021/acs.biochem.4c00502","url":null,"abstract":"<p><p>Transmembrane glucose transport, facilitated by glucose transporters (GLUTs), is commonly understood through the simple mobile carrier model (SMCM), which suggests that the central binding site alternates exposure between the inside and outside of the cell, facilitating glucose exchange. An alternative \"multisite model\" posits that glucose transport is a stochastic diffusion process between ligand-operated gates within the transporter's central channel. This study aims to test these models by conducting atomistic molecular dynamics simulations of multiple glucose molecules docked along the central cleft of GLUT1 at temperatures both above and below the lipid bilayer melting point. Our results show that glucose exchanges occur on a nanosecond time-scale as glucopyranose rings slide past each other within the channel cavities, with minimal protein conformational movement. While bilayer gelation slows net glucose transit, the frequency of positional exchanges remains consistent across both temperatures. This supports the observation that glucose exchange at 0 °C is much faster than net flux, aligning with experimental data that show approximately 100 times the rate of exchange flux relative to net flux at 0 °C compared to 37 °C.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"928-939"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11840925/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057514","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}

{"title":"Kinetics of the Oxidation of the [2Fe-2S] Cluster in SoxR by Redox-Active Compounds as Studied by Pulse Radiolysis.","authors":"Kazuo Kobayashi, Takahiro Tanaka, Takahiro Kozawa","doi":"10.1021/acs.biochem.4c00679","DOIUrl":"10.1021/acs.biochem.4c00679","url":null,"abstract":"<p><p>SoxR containing a [2Fe-2S] cluster required for its transcription activity functions as a bacterial stress-response sensor that is activated through oxidation by redox-active compounds (RACs). SoxR from <i>Escherichia coli</i> (EcSoxR) is activated by nearly all RACs nonspecifically. In contrast, nonenteric SoxRs such as <i>Pseudomonas aeruginosa</i> (PaSoxR), and <i>Streptomyces coelicolor</i> (ScSoxR) activate their target genes in response to RAC including endogenously produced metabolites. To investigate the determinants of SoxR's activity, the endogenous or various synthetic RACs-mediated oxidation of the [2Fe-2S] cluster of EcSoxR, PaSoxR, and ScSoxR were measured by pulse radiolysis. Radiolytically generated hydrated electrons (e_aq^-) very rapidly reduced the oxidized form of the [2Fe-2S] cluster of SoxR. In the presence of RAC, a subsequent increase in absorption in the visible region corresponding to reoxidation of the [2Fe-2S] cluster was observed on a time scale of milliseconds. Both EcSoxR and PaSoxR reacted very rapidly (2.0 × 10⁸ to 2.0 × 10⁹ M^-1 s^-1) with various RACs, including viologen, phenazines, and quinones. No differences in kinetic behaviors were evident between EcSoxR and PaSoxR, whereas ScSoxR reacted with a limited range of RACs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"895-902"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062156","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}

{"title":"Molecular Glues: A New Approach to Modulating GPCR Signaling Bias.","authors":"Jamie Kushnir, Ryan H Gumpper","doi":"10.1021/acs.biochem.4c00734","DOIUrl":"10.1021/acs.biochem.4c00734","url":null,"abstract":"<p><p>G-protein-coupled receptors (GPCRs) transmit an extracellular chemical/biological signal across the cell membrane, stimulating an array of intracellular signaling cascades. Canonically, these extracellular signaling molecules bind to the endogenous ligand pocket (orthosteric pocket), which stabilizes either an active or inactive conformational ensemble of the receptor. However, recent structural evidence indicates that small molecules can mediate the protein-protein interactions between the GPCR and their intracellular transducers. These small molecules are reminiscent of molecular glues and can be powerful tools for modulating GPCR signaling bias. In this Perspective, we will investigate the current structural information available on molecular glues and how they modulate GPCR signaling bias. We also examine the prospects of molecular glues and GPCR drug/probe design.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"749-759"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11840928/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121671","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}

{"title":"A Cascade of Conformational Switches in SARS-CoV-2 Frameshifting: Coregulation by Upstream and Downstream Elements.","authors":"Samuel Lee, Shuting Yan, Abhishek Dey, Alain Laederach, Tamar Schlick","doi":"10.1021/acs.biochem.4c00641","DOIUrl":"10.1021/acs.biochem.4c00641","url":null,"abstract":"<p><p>Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against COVID-19. In this process, a -1 shift in the ribosomal reading frame encodes alternative viral proteins. Any interference with this process profoundly affects viral replication and propagation. For SARS-CoV-2, two RNA sites associated with ribosomal frameshifting are positioned on the 5' and 3' of the frameshifting residues. Although much attention has been focused on the 3' frameshift element (FSE), the 5' stem-loop (attenuator hairpin, AH) can play a role. Yet the relationship between the two regions is unknown. In addition, multiple folds of the FSE and FSE-containing RNA regions have been discovered. To gain more insight into these RNA folds in the larger sequence context that includes AH, we apply our graph-theory-based modeling tools to represent RNA secondary structures, \"RAG\" (RNA-As-Graphs), to generate conformational landscapes that suggest length-dependent conformational distributions. We show that the AH region can coexist as a stem-loop with main and alternative 3-stem pseudoknots of the FSE (dual graphs 3_6 and 3_3 in our notation) but that an alternative stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. A critical length for AS1 of 10-bp regulates key folding transitions. Together with designed mutants and available experimental data, we present a sequential view of length-dependent folds during frameshifting and suggest their mechanistic roles. These structural and mutational insights into both ends of the FSE advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting how alternative folds play a role in frameshifting and defining potential therapeutic intervention techniques that target specific folds.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"953-966"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11840926/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187548","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}

{"title":"Kinetic and Thermodynamic Characterization of Human 4-Oxo-l-proline Reductase Catalysis.","authors":"Ennio Pečaver, Greice M Zickuhr, Teresa F G Machado, David J Harrison, Rafael G da Silva","doi":"10.1021/acs.biochem.4c00721","DOIUrl":"10.1021/acs.biochem.4c00721","url":null,"abstract":"<p><p>The enzyme 4-oxo-l-proline reductase (BDH2) has recently been identified in humans. BDH2, previously thought to be a cytosolic (<i>R</i>)-3-hydroxybutyrate dehydrogenase, actually catalyzes the NADH-dependent reduction of 4-oxo-l-proline to <i>cis</i>-4-hydroxy-l-proline, a compound with known anticancer activity. Here we provide an initial mechanistic characterization of the BDH2-catalyzed reaction. Haldane relationships show the reaction equilibrium strongly favors the formation of <i>cis</i>-4-hydroxy-l-proline. Stereospecific deuteration of NADH C4 coupled with mass spectrometry analysis of the reaction established that the pro<i>-S</i> hydrogen is transferred. NADH is co-purified with the enzyme, and a binding kinetics competition assays with NAD⁺ defined dissociation rate constants for NADH of 0.13 s^-1 at 5 °C and 7.2 s^-1 at 25 °C. Isothermal titration calorimetry at 25 °C defined equilibrium dissociation constants of 0.48 and 29 μM for the BDH2:NADH and BDH2:NAD⁺ complexes, respectively. Differential scanning fluorimetry showed BDH2 is highly thermostabilized by NADH and NAD⁺. The <i>k</i>_cat/<i>K</i>_M pH-rate profile indicates that a group with a p<i>K</i>_a of 7.3 and possibly another with a p<i>K</i>_a of 8.7 must be deprotonated and protonated, respectively, for maximum binding of 4-oxo-l-proline and/or catalysis, while the <i>k</i>_cat profile is largely insensitive to pH in the pH range used. The single-turnover rate constant is only 2-fold higher than <i>k</i>_cat. This agrees with a pre-steady-state burst of substrate consumption, suggesting that a step after chemistry, possibly product release, contributes to limit <i>k</i>_cat. A modest solvent viscosity effect on <i>k</i>_cat indicates that this step is only partially diffusional. Taken together, these data suggest chemistry does not limit the reaction rate but may contribute to it.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"860-870"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11840923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062188","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}

{"title":"Conversion of Inactive Non-Pro1 Tautomerase Superfamily Members into Active Tautomerases: Analysis of the Pro1 Mutants.","authors":"Emily B Lancaster, Haley A Hardtke, Trevor R Melkonian, Mukesh Venkat Ramani, William H Johnson, Bert-Jan Baas, Y Jessie Zhang, Christian P Whitman","doi":"10.1021/acs.biochem.4c00338","DOIUrl":"10.1021/acs.biochem.4c00338","url":null,"abstract":"<p><p>Pro1 is a critical catalytic residue in the characterized activities of tautomerase superfamily (TSF) members. Only a handful of members (∼346) lack Pro1 in a sequence similarity network (SSN) that consists of over 11,000 members. Most (294 members) are in the malonate semialdehyde decarboxylase (MSAD)-like subgroup, but the ones characterized thus far have little or no MSAD activity. Moreover, there is little to no activity with other TSF substrates. Five non-Pro1 members were selected randomly for kinetic [using phenylenolpyruvate (PP) and 2-hydroxymuconate (2HM)], mutagenic, inhibition, and crystallographic analysis. Using PP, <i>k</i>_cat/<i>K</i>_m values (∼10¹-10² M^-1 s^-1) could be estimated for three native proteins whereas using 2HM, a <i>k</i>_cat/<i>K</i>_m value could only be estimated for one native protein (∼10³ M^-1 s^-1). The <i>k</i>_cat and <i>K</i>_m values could not be determined. However, changing the N-terminal residue to a proline gave a significant improvement in <i>k</i>_cat/<i>K</i>_m values for all mutant enzymes using PP or 2HM. For PP, the <i>k</i>_cat/<i>K</i>_m values ranged from 10³-10⁵ M^-1 s^-1 and for 2HM, the <i>k</i>_cat/<i>K</i>_m values ranged from 10²-10⁴ M^-1 s^-1. In addition, it was now possible to measure <i>k</i>_cat and <i>K</i>_m values for all mutant proteins using PP and one mutant protein using 2HM. Incubation of the Pro1 mutants with 3-bromopropiolate (3BP) results in covalent modification of the prolyl nitrogen of Pro1 by a 3-oxopropanoate adduct. Crystallographic analysis of two mutant enzymes (NJ7V1P and 8U6S1P) modified by the 3-oxopropanoate adduct identified binding ligands and suggest a mechanism for the tautomerase activity involving Pro1, Arg71, Tyr124, and the backbone amide of Phe68.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"812-822"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363269","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}

{"title":"Pathway Specific Unbinding Free Energy Profiles of Ritonavir Dissociation from HIV-1 Protease.","authors":"Emily Vig, Jianan Sun, Chia-En A Chang","doi":"10.1021/acs.biochem.4c00560","DOIUrl":"10.1021/acs.biochem.4c00560","url":null,"abstract":"<p><p>Investigation of protein-drug recognition is key to understanding drug selectivity and binding affinity. In combination, the binding/unbinding free energy landscape and intermolecular interactions can be used to understand drug binding/unbinding mechanisms. This information is vital for the development of drugs with improved efficacy and explanation of mutation effects. This study investigated the dissociation processes of ritonavir unbinding from HIV protease (HIVp). Analyzing unbinding trajectories modeled by accelerated molecular dynamics (MD) simulations, three distinct pathways, pathways A-C, were characterized. Using a reduced dimensionality strategy with the principal component analysis, we carried out short classical MD runs with explicit water to sample local fluctuation during ritonavir dissociation and applied the milestoning theory to construct an unbinding free energy landscape. We found that each pathway showed similar values of binding free energy, albeit pathway A accounts for over 50% of dissociation trajectories. Interestingly, residue-residue correlation network analysis showed that in pathway A, a broad correlation network outside the flap region governs protein motions during ritonavir unbinding, which includes residues with reported mutation effects. However, the other two pathways showed limited correlation networks where no reported mutated residues were involved, explaining the favorability of pathway A. Guided by the free energy profile, we investigated each energy barrier and minimum, demonstrating that hydrogen bonding governed movement of the flap regions, directly impacting the calculated energy. Our study provided a new strategy to estimate ligand binding free energy and demonstrated the importance of the transient interactions during ligand-protein dissociation pathways in understanding drug unbinding.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"940-952"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11844232/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143381263","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}

{"title":"Redox-Guided DNA Scanning by the Dynamic Repair Enzyme Endonuclease III.","authors":"Ayaz Hassan, Filipe C D A Lima, Frank N Crespilho","doi":"10.1021/acs.biochem.4c00621","DOIUrl":"10.1021/acs.biochem.4c00621","url":null,"abstract":"<p><p>Endonuclease III (EndoIII), a key enzyme in the base excision repair (BER) pathway, contains a [4Fe4S] cluster that facilitates DNA repair through DNA-mediated charge transfer. Recent findings indicate that the redox state of this cluster influences EndoIII's binding affinity for DNA, modulating the enzyme's activity. In this study, we investigated the structural and electronic changes of the [4Fe4S] cluster upon binding to double-stranded DNA (dsDNA) using Fourier transform infrared spectroscopy, density functional theory calculations, and machine learning models. Our results reveal shifts in Fe-S bond vibrational modes, suggesting stabilization of the oxidized [4Fe4S] cluster in proximity to negatively charged DNA. A machine learning model, trained on the spectral features of the EndoIII/DNA complex, predicted the enzyme-DNA binding distance, providing further insights into the structural changes upon binding. We correlated the electrochemical stabilization potential of 150 mV in the [4Fe4S] cluster with the enzyme's DNA-binding properties, demonstrating how the cluster's redox state plays a crucial role in both structural stability and DNA repair.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"782-790"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11840932/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187574","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}

{"title":"Transcription Regulation of Flagellins: A Structural Perspective.","authors":"Sheenu, Deepti Jain","doi":"10.1021/acs.biochem.4c00791","DOIUrl":"10.1021/acs.biochem.4c00791","url":null,"abstract":"<p><p>Bacterial flagella are complex molecular motors that are essential for locomotion and host colonization. They consist of 30 different proteins expressed in varying stoichiometries. Their assembly and function are governed by a hierarchical transcriptional regulatory network with multiple checkpoints primarily regulated by sigma factors. Expression of late flagellar genes requires the complete assembly of the flagellar basal body and hook. The extracellular segment of the flagellum, termed filament, is composed of self-assembling flagellin subunits encoded by the <i>fliC</i> gene and harbors potent antigenic epitopes. Structural studies have illuminated the molecular mechanisms underlying its assembly and its regulation at the transcription level. σ²⁸, a key subunit of the RNA polymerase complex, binds to specific promoter sequences to initiate transcription of late flagellar genes, while its activity is controlled by the antisigma factor FlgM. This review summarizes current insights into the structural characterization of flagellins across various bacterial species, their transcription by σ²⁸, and the structural mechanism controlling σ²⁸ activity through FlgM. Additionally, we highlight the regulation of flagellin gene expression via transcription factors and their post-transcriptional regulation, providing a comprehensive overview of the intricate mechanisms that support bacterial motility and adaptation.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"770-781"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057518","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}

{"title":"Alternative Role of B/b Knob-Hole Interactions in the Fibrin Assembly.","authors":"Tatyana Platonova, Oleksii Hrabovskyi, Volodymyr Chernyshenko, Yevhenii Stohnii, Yevhenii Kucheriavyi, Kateryna Baidakova, Daria Korolova, Anna Urbanowicz, Serhiy Komisarenko","doi":"10.1021/acs.biochem.4c00695","DOIUrl":"10.1021/acs.biochem.4c00695","url":null,"abstract":"<p><p>The self-assembly of fibrin is a vital process in blood clotting, primarily facilitated by the interactions between knobs \"A\" and \"B\" in the central E region of one molecule and the corresponding holes \"a\" and \"b\" in the peripheral D regions of two other fibrin molecules. However, the precise function of the interactions between knob \"B\" and hole \"b\" during fibrin polymerization remains a subject of ongoing debate. The present study focuses on investigating intermolecular interactions between knob \"B\" and hole \"b\". We investigated the D-E-D interactions within the fibrin protofibril to accomplish this objective. Our investigation involved studying the formation of supramolecular complexes involving desAB fibrin with fibrin(ogen) fragments, specifically the D-dimer and D fragment. The research utilized analytical size-exclusion chromatography, SDS-PAGE and densitometry of SDS-PAGE images, dynamic light scattering measurements, turbidity studies, electron microscopy, and computer modeling. Our findings indicate that the interference of the D fragment into classical D-E-D interaction occurs through knob \"B\" of the fibrin molecule. Molecular dynamics simulations elucidate the binding of only one D region, attributed to the shift of the D-dimer toward the fibrin desAB molecule. The formation of such a complex can be considered evidence supporting the potential mechanism of the branching of protofibrils. According to this theoretical mechanism, the inclusion of the D region from an external fibrin molecule into D-E-D interactions is facilitated through \"B/b\" contacts.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"791-800"},"PeriodicalIF":2.9,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143044909","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}