Biochemistry Biochemistry最新文献

{"title":"Redox-Guided DNA Scanning by the Dynamic Repair Enzyme Endonuclease III","authors":"Ayaz Hassan,&nbsp;Filipe C. D. A. Lima and Frank N. Crespilho*,&nbsp;","doi":"10.1021/acs.biochem.4c0062110.1021/acs.biochem.4c00621","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00621https://doi.org/10.1021/acs.biochem.4c00621","url":null,"abstract":"<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":"64 4","pages":"782–790 782–790"},"PeriodicalIF":2.9,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00621","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428603","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":"Molecular Glues: A New Approach to Modulating GPCR Signaling Bias","authors":"Jamie Kushnir,&nbsp; and ,&nbsp;Ryan H. Gumpper*,&nbsp;","doi":"10.1021/acs.biochem.4c0073410.1021/acs.biochem.4c00734","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00734https://doi.org/10.1021/acs.biochem.4c00734","url":null,"abstract":"<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":"64 4","pages":"749–759 749–759"},"PeriodicalIF":2.9,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00734","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428592","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":"Identifying Allosteric Hotspots in Mycobacterium tuberculosis cAMP Receptor Protein through Structural Homology","authors":"Stephen P. Dokas,&nbsp;Daniel K. Taylor,&nbsp;Lydia L. Good,&nbsp;Sanuja Mohanaraj and Rodrigo A. Maillard*,&nbsp;","doi":"10.1021/acs.biochem.4c0072310.1021/acs.biochem.4c00723","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00723https://doi.org/10.1021/acs.biochem.4c00723","url":null,"abstract":"<p >Understanding the mechanisms of allosteric regulation in response to second messengers is crucial for advancing basic and applied research. This study focuses on the differential allosteric regulation by the ubiquitous signaling molecule, cAMP, in the cAMP receptor protein from <i>Escherichia coli</i> (CRP_Ecoli) and from <i>Mycobacterium tuberculosis</i> (CRP_MTB). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRP_Ecoli into CRP_MTB and examining their effects on protein solution structure, stability and function, we aimed to determine the factors contributing to their differential allosteric regulation. Our results demonstrate that the mutations did not significantly alter the overall fold, assembly and thermodynamic stability of CRP_MTB, but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRP_MTB to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRP_MTB–DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRP_MTB–DNA complexes serve as a nucleation point for additional binding of CRP_MTB proteins to form high-order oligomers with the DNA. Overall, our findings highlight the importance of both cAMP and DNA interactions in modulating the allosteric regulation of CRP_MTB and provide insights into the differential responses of CRP_Ecoli and CRP_MTB to cAMP.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 4","pages":"801–811 801–811"},"PeriodicalIF":2.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00723","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428484","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,&nbsp;Greice M. Zickuhr,&nbsp;Teresa F. G. Machado,&nbsp;David J. Harrison and Rafael G. da Silva*,&nbsp;","doi":"10.1021/acs.biochem.4c0072110.1021/acs.biochem.4c00721","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00721https://doi.org/10.1021/acs.biochem.4c00721","url":null,"abstract":"<p >The enzyme 4-oxo-<span>l</span>-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-<span>l</span>-proline to <i>cis</i>-4-hydroxy-<span>l</span>-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-<span>l</span>-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-<span>l</span>-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":"64 4","pages":"860–870 860–870"},"PeriodicalIF":2.9,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00721","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428419","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":"Structural and Mechanistic Insights into the Main Protease (Mpro) Dimer Interface Destabilization Inhibitor: Unveiling New Therapeutic Avenues against SARS-CoV-2.","authors":"Ankur Singh, Kuldeep Jangid, Sanketkumar Nehul, Preeti Dhaka, Ruchi Rani, Akshay Pareek, Gaurav Kumar Sharma, Pravindra Kumar, Shailly Tomar","doi":"10.1021/acs.biochem.4c00535","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00535","url":null,"abstract":"<p><p>SARS-CoV-2 variant recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral targets. Mpro features several druggable sites, including active sites and allosteric sites near the dimerization interface, that regulate its catalytic activity. This study identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to the target Mpro was validated. The FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC₅₀ values in the range from 0.64 to 11.98 μM. Additionally, <i>in vitro</i> cell-based antiviral assays showed high efficacy with EC₅₀ values in the range of 1.51 to 18.92 μM. The crystal structure of the Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for the proteolytic activity of Mpro. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and direct the rational design of minocycline derivatives as antiviral drugs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062210","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":"Kinetics of the Oxidation of the [2Fe-2S] Cluster in SoxR by Redox-Active Compounds as Studied by Pulse Radiolysis","authors":"Kazuo Kobayashi*,&nbsp;Takahiro Tanaka and Takahiro Kozawa,&nbsp;","doi":"10.1021/acs.biochem.4c0067910.1021/acs.biochem.4c00679","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00679https://doi.org/10.1021/acs.biochem.4c00679","url":null,"abstract":"<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":"64 4","pages":"895–902 895–902"},"PeriodicalIF":2.9,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428417","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":"Transcription Regulation of Flagellins: A Structural Perspective","authors":"Sheenu,&nbsp; and ,&nbsp;Deepti Jain*,&nbsp;","doi":"10.1021/acs.biochem.4c0079110.1021/acs.biochem.4c00791","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00791https://doi.org/10.1021/acs.biochem.4c00791","url":null,"abstract":"<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":"64 4","pages":"770–781 770–781"},"PeriodicalIF":2.9,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428372","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":"Insight into the Mechanism of d-Glucose Accelerated Exchange in GLUT1 from Molecular Dynamics Simulations","authors":"Carmen Domene*,&nbsp;Brian Wiley,&nbsp;Saul Gonzalez-Resines and Richard J. Naftalin*,&nbsp;","doi":"10.1021/acs.biochem.4c0050210.1021/acs.biochem.4c00502","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00502https://doi.org/10.1021/acs.biochem.4c00502","url":null,"abstract":"<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":"64 4","pages":"928–939 928–939"},"PeriodicalIF":2.9,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00502","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428420","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":"Carboxy-Amidated AamAP1-Lys has Superior Conformational Flexibility and Accelerated Killing of Gram-Negative Bacteria","authors":"Rosalind J. Van Wyk,&nbsp;June C. Serem,&nbsp;Carel B. Oosthuizen,&nbsp;Dorothy Semenya,&nbsp;Miruna Serian,&nbsp;Christian D. Lorenz,&nbsp;A. James Mason*,&nbsp;Megan J. Bester and Anabella R. M. Gaspar*,&nbsp;","doi":"10.1021/acs.biochem.4c0058010.1021/acs.biochem.4c00580","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00580https://doi.org/10.1021/acs.biochem.4c00580","url":null,"abstract":"<p >C-terminal amidation of antimicrobial peptides (AMPs) is a frequent minor modification used to improve antibacterial potency, commonly ascribed to increased positive charge, protection from proteases, and a stabilized secondary structure. Although the activity of AMPs is primarily associated with the ability to penetrate bacterial membranes, hitherto the effect of amidation on this interaction has not been understood in detail. Here, we show that amidation of the scorpion-derived membranolytic peptide AamAP1-Lys produces a potent analog with faster bactericidal activity, increased membrane permeabilization, and greater Gram-negative membrane penetration associated with greater conformational flexibility. AamAP1-lys-NH₂ has improved antibiofilm activity against <i>Acinetobacter baumannii</i> and <i>Escherichia coli</i>, benefits from a two- to 3-fold selectivity improvement, and provides protection against <i>A. baumannii</i> infection in a <i>Galleria mellonella</i> burn wound model. Circular dichroism spectroscopy shows both peptides adopt α-helix conformations in the steady state. However, molecular dynamics (MD) simulations reveal that, during initial binding, AamAP1-Lys-NH₂ has greater conformation heterogeneity, with substantial polyproline-II conformation detected alongside α-helix, and penetrates the bilayer more readily than AamAP1-Lys. AamAP1-Lys-NH₂ induced membrane permeabilization of <i>A. baumannii</i> occurs only above a critical concentration with slow and weak permeabilization and slow killing occurring at its lower MIC but causes greater and faster permeabilization than AamAP1-Lys, and kills more rapidly, when applied at equal concentrations. Therefore, while the increased potency of AamAP1-Lys-NH₂ is associated with slow bactericidal killing, amidation, and the conformational flexibility it induces, affords an improvement in the AMP pharmacodynamic profile and may need to be considered to achieve improved therapeutic performance.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 4","pages":"841–859 841–859"},"PeriodicalIF":2.9,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00580","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428421","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":"Mechanistic Cooperation of the Two Pore-Forming Transmembrane Motifs Regulates the β-Barrel Pore Formation by Listeriolysin O","authors":"Kusum Lata,&nbsp;Koyel Nandy,&nbsp; Geetika and Kausik Chattopadhyay*,&nbsp;","doi":"10.1021/acs.biochem.4c0059210.1021/acs.biochem.4c00592","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00592https://doi.org/10.1021/acs.biochem.4c00592","url":null,"abstract":"<p >Listeriolysin O (LLO) is a potent membrane-damaging pore-forming toxin (PFT) secreted by the bacterial pathogen <i>Listeria monocytogenes</i>. LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), which specifically target cholesterol-containing cell membranes to form oligomeric pores and induce membrane damage. CDCs, including LLO, harbor designated pore-forming motifs. In the soluble monomeric state, these motifs are present as helical segments (two transmembrane helices (TMHs); TMH1 and TMH2), and in the course of oligomeric pore formation, they convert into transmembrane β-hairpins to form the β-barrel scaffold of the CDC pores. Despite their well-established role in forming the β-barrel pore scaffold, precise structural implications of the two distinct TMH motifs and their membrane-insertion mechanism still remain obscure. Here, we show that the two TMH motifs of LLO contribute differently to maintaining the structural integrity of the toxin. While the deletion of TMH1 imposed a more serious defect, truncation of TMH2 was found to have a less severe effect on the structural integrity. Despite showing membrane-binding and oligomerization ability, the TMH2-deleted LLO variant displayed drastically abrogated pore-forming activity, presumably due to compromised membrane-insertion efficacy of the pore-forming TMH motifs. When probed for the membrane-insertion mechanism, we found slower membrane-insertion kinetics for TMH2 than for TMH1. Interestingly, deletion of TMH2 arrested membrane insertion of TMH1, thus suggesting a stringent cooperation between the two TMH motifs in regulating the pore-formation mechanism of LLO. Taken together, our study provides new mechanistic insights regarding the membrane-damaging action of LLO, in the CDC family of PFTs.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 4","pages":"917–927 917–927"},"PeriodicalIF":2.9,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143428360","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}