Biochemistry Biochemistry最新文献

{"title":"Stereochemistry and Charged State Influence Effector Outcomes of d-2-Hydroxyglutarate Dehydrogenase Ligands.","authors":"Joanna Afokai Quaye, Giovanni Gadda","doi":"10.1021/acs.biochem.5c00408","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00408","url":null,"abstract":"<p><p>d-2-Hydroxyglutarate dehydrogenase (D2HGDH) has recently received considerable attention due to the involvement of d-2-hydroxyglutarate in various medical conditions. This enzyme has been reported to diverge in substrate scope depending on whether its source is prokaryotic or eukaryotic. The D2HGDH from <i>Pseudomonas aeruginosa</i>, <i>Pa</i>D2HGDH, is of particular interest due to its requirement for <i>P. aeruginosa</i> survival via the l-serine biosynthesis pathway and its potential use as a therapeutic target against the bacterium. The enzyme, which is active on d-2-hydroxyglutarate (D2HG) and d-malate, is a Zn²⁺- and FAD-dependent dehydrogenase that employs metal-triggered flavin reduction in its catalytic mechanism. While <i>Pa</i>D2HGDH is the most extensively studied D2HGDH homologue, no studies have investigated the ligand-binding modalities in the enzyme, and─for that matter─any D2HGDH homologue. This study investigated the inhibition profiles of <i>Pa</i>D2HGDH by various D2HG and d-malate analogues. The study demonstrates that stereochemistry and functional groups at the C2 position of ligands are key determinants of binding to <i>Pa</i>D2HGDH. The enzyme recognizes d-isomeric ligands as substrates, with l-isomers acting as reversible inhibitors. Ligand binding requires bidentate coordination with the active site Zn²⁺ cofactor, with longer chain ligands and polar ligands having lower <i>K</i>_is and Δ<i>G</i>^o values due to enhanced interactions with the highly polar active site. Hydrophobic and van der Waals interactions also contribute to ligand binding in <i>Pa</i>D2HGDH. The study concludes that <i>Pa</i>D2HGDH can be reversibly inhibited, providing a foundation for biochemical studies on <i>Pa</i>D2HGDH inhibitors, with direct applications to D2HG biosensor development.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022489","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":"Electron Pushing: Variants That Alter the Catalytic Distribution of Electrons in Dihydroorotate Dehydrogenase 1B from Lactococcus lactis","authors":"Corine O. Smith,&nbsp;Richa Khatiwada,&nbsp;Pengfei Li and Graham R. Moran*,&nbsp;","doi":"10.1021/acs.biochem.5c00409","DOIUrl":"10.1021/acs.biochem.5c00409","url":null,"abstract":"<p >Dihydroorotate dehydrogenase 1B (DHOD1B) is one of several flavoproteins that utilize active half-sites. These enzymes have two flavin cofactors (FAD and FMN) that each interact with a specific reductant/oxidant substrate/product. Electrons gained at one-half-site must be transmitted to the other half-site and iron–sulfur centers between the flavin cofactors serve in this role. DHOD1B from <i>Lactococcus lactis</i> (LlDHOD1B) is a heterodimeric protein that has been shown to fractionally accumulate a flavin bisemiquinone state comprised of equimolar anionic and neutral forms, demonstrating an internal electron distribution equilibrium. Variant forms of LlDHOD1B were designed to perturb predicted or claimed pathways for internal transmission of electrons. Lysine 48 of the PyrD subunit is positioned near the FMN isoalloxazine N5 and the orotate C6-carboxylate. The K48M variant revealed that the anionic semiquinone resides at the FMN cofactor and that the lysine’s role is electrostatic, influencing both the p<i>K</i>_a and reduction potential of the FMN. Glutamate 221 of the PyrK subunit stacks with the FAD isoalloxazine. The E221Q variant established that this charge influences the rate of hydride transfer from NADH and the rate of reduction of orotate and accumulates little of the flavin bisemiquinone observed with the WT enzyme. Cysteine 135 of the PyrD subunit serves as the active half-site acid/base. The C135A variant prevented reduction of orotate, permitting the influence of orotate binding on the reduction potential of the FMN cofactor to be determined indicating a +70 mV change in the FMN reduction potential with the association of orotate.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3971–3985"},"PeriodicalIF":3.0,"publicationDate":"2025-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145013452","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":"ssDNA and ssRNA Promote Phase Condensation of SAMHD1","authors":"Brandon E. Smith,&nbsp;Ankita Pohnerkar,&nbsp;Benjamin Orris,&nbsp;Shridhar Bhat,&nbsp;Matthew Egleston and James T. Stivers*,&nbsp;","doi":"10.1021/acs.biochem.5c00422","DOIUrl":"10.1021/acs.biochem.5c00422","url":null,"abstract":"<p >SAMHD1 (SAM domain and HD domain-containing protein 1) is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) with functions in viral restriction, R-loop resolution, DNA repair, telomere maintenance, ssRNA homeostasis, and regulation of self-nucleic acids. As a dNTPase, SAMHD1 functions as an allosterically activated tetramer, where binding of GTP to the A1 activator site of each monomer initiates dNTP-dependent tetramerization. cEM structures reveal that the nucleic-acid-related functions of SAMHD1 involve binding of guanine residues to the A1 site, leading to oligomeric forms that appear as beads-on-a-string on single-stranded RNA and DNA. SAMHD1’s cellular activities and known protein interactions involve liquid–liquid phase separation (LLPS), although there are no reports that SAMHD1 itself exhibits phase separation properties. The protein phase separation prediction algorithm MolPhase indicated an overall phase separation probability score of 0.65 and suggested that the amino terminal SAM domain and the disordered carboxyl terminus (CT) may promote phase separation. Although no phase separation behavior was observed in physiological buffer, in the presence of 9% PEG 2000 and ssDNA or ssRNA, SAMHD1 condensed into liquid-like droplets. These droplets were disrupted by deletion of the SAM or CT domains, showed fusion behavior, and were rapidly disrupted by the addition of A1 site ligands GTP, dGTP, and small-molecule inhibitors. We also observed that SAMHD1-ssDNA condensates within the nuclei of human cells in microinjection experiments, supporting a biological relevance for such complexes. LLPS by SAMHD1 could serve a regulatory role in cells and provide a new therapeutic target for the treatment of cancer and viral infections.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3886–3900"},"PeriodicalIF":3.0,"publicationDate":"2025-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145005676","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":"Structural Elements of Dwarf Open Reading Frame Required for Activation of the Sarco-Endoplasmic Reticulum Calcium Pump","authors":"M’Lynn E. Fisher,&nbsp;Justin R. Gregory,&nbsp;Stan T. J. Aanhane,&nbsp;M. Joanne Lemieux and Howard S. Young*,&nbsp;","doi":"10.1021/acs.biochem.5c00305","DOIUrl":"10.1021/acs.biochem.5c00305","url":null,"abstract":"<p >The sarco-endoplasmic reticulum calcium pump (SERCA) is a P-type ATPase that plays a critical role in intracellular calcium signaling. SERCA maintains the calcium gradient between the cytosol and the sarco-endoplasmic reticulum, which is essential for a variety of physiological events including the muscle contraction-relaxation cycle. In cardiac muscle, SERCA is regulated by transmembrane peptides phospholamban (PLN) and dwarf open reading frame (DWORF). These peptides encode the opposing functions of SERCA inhibition by PLN and SERCA activation by DWORF, though the underlying mechanisms remain unclear. Herein, we investigated structural elements of DWORF expected to play a role in SERCA activation. We first measured SERCA activity in the absence and presence of DWORF variants targeting Leu¹² and Pro¹⁵. These residues were selected based on sequence alignment with PLN. Leu¹² and Pro¹⁵ of DWORF align with the essential residues Leu³¹ and Asn³⁴ of PLN, which are required for SERCA inhibition. We found that both residues are required for SERCA activation by DWORF and that substitution of Pro¹⁵ (to Ala, Asn, or Leu) resulted in potent inhibition of SERCA. We next investigated the roles of Gly²¹, Ile²³, and Gly²⁵ in SERCA activation and DWORF oligomerization. These residues are part of a common helix interaction motif, GxxxG (Gly²¹-Trp-Ile-Val-Gly²⁵) found in DWORF, which is unique among the regulins. The data suggest that the GxxxG motif does not play a role in DWORF oligomerization. Instead, this motif appears to interact with SERCA and provides a smooth interface that promotes activation and avoids inhibitory interactions with SERCA.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"4000–4010"},"PeriodicalIF":3.0,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144999276","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":"Domain-Specific DNA Binding Activities of BRCA1 Reveal Substrate Preferences for Homologous Recombination and Telomere Regulation","authors":"Kaitlin Lowran,&nbsp;Laura Campbell,&nbsp;Emma Cismas and Colin G. Wu*,&nbsp;","doi":"10.1021/acs.biochem.5c00333","DOIUrl":"10.1021/acs.biochem.5c00333","url":null,"abstract":"<p >BRCA1 is a crucial component of homologous recombination (HR), a high-fidelity pathway for repairing double-stranded DNA breaks (DSBs) in human cells. The central region of the BRCA1 protein contains two putative DNA binding domains (DBDs), yet their relative substrate specificities and functional contributions to HR remain unclear. Here, we characterized the DNA binding properties of DBD1 (amino acids 330–554), DBD2 (amino acids 894–1057), and BRCA1 C-terminal (BRCT) repeats using biolayer interferometry. Affinities were determined for single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex (G4) DNA. DBD2 exhibited strong and nearly identical binding to all three substrates (<i>K</i>_d = ∼35–44 nM), while the BRCT also bound to each structure similarly, but with lower affinity (<i>K</i>_d = ∼149–184 nM). In contrast, DBD1 showed a distinct preference for dsDNA, binding approximately 2-fold tighter compared to ssDNA or G4. These findings support a model in which BRCA1 uses modular DNA binding domains to recognize diverse repair targets; DBD2 serves as a primary anchor to associate with a broad range of DNA structures with BRCT contributing to the contacts. DBD1 acts as the determinant of DNA structure-specific localization that may help direct BRCA1 to DSB sites during HR or to noncanonical elements such as chromatin and telomeres. These insights lay the groundwork for future studies examining how cancer-associated variants affect the DNA binding and repair phenotypes of BRCA1 and may inform the interpretation of variants of unknown clinical significance.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3819–3828"},"PeriodicalIF":3.0,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445000/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144990966","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 Single Mutation in the “DSL” Motif of the Acyl Carrier Protein Can Prevent Its In Vivo Phosphopantetheinylation by E. coli Holo-Acyl Carrier Protein Synthase (AcpS)","authors":"Chetna Dhembla,&nbsp;Debodyuti Sadhukhan,&nbsp;Rashima Prem,&nbsp;Shivangi Vaish,&nbsp;Shalini Verma,&nbsp;Suman Kundu and Monica Sundd*,&nbsp;","doi":"10.1021/acs.biochem.4c00822","DOIUrl":"10.1021/acs.biochem.4c00822","url":null,"abstract":"<p >The <i>Escherichia coli</i> expression system is the method of choice to obtain high yields of a pure protein. Since most biological pathways are evolutionarily conserved from bacteria to mammals, there is always a chance that a non-native protein shares sequence or structural homology with the natural substrate of an <i>E. coli</i> enzyme. In such cases, when this foreign protein is overexpressed in <i>E. coli</i>, it may be processed as a substrate by that enzyme, resulting in its modification. A notable example is the heterologous expression of Type II acyl carrier proteins (ACPs) in <i>E. coli</i>. Due to the conservation of a type II fatty acid synthesis pathway (FAS) across bacteria to mammals, the non-native type II ACPs are often recognized as a substrate by the <i>E. coli</i> 4′-phosphopantetheinyl transferase, also known as the Holo-acyl carrier protein synthase (AcpS). This undesirable modification is a concern when the objective is to obtain milligram amounts of apo-ACP. Here, using an approach combining mutagenesis, enzyme activity, and NMR, we have probed for the <i>E. coli</i> ACP (AcpP) residues that can prevent this <i>in vivo</i> modification. Taking cues from the AcpP–AcpS crystal structure (PDB entry 1F80), five charge-neutralization mutations were designed on the AcpP surface, i.e., D35N, E41A, E47A, E48A, and E47A/E48A, to disrupt the AcpP–AcpS interaction. All the AcpP mutants except D35N expressed as partially phosphopantetheinylated proteins in <i>E. coli</i>, presenting D35N mutagenesis as an attractive approach to prevent undesired modification of AcpP <i>in vivo.</i> The strategy was tested on two other non-native type II ACPs that express predominantly as phosphopantetheinylated proteins in <i>E. coli</i>, <i>Mus musculus</i> mitochondrial FAS ACP (mACP), and <i>Salmonella</i> Typhimurium invasion acyl carrier protein (IacP). A single D35N mutation in the “DSL” motif of these ACPs prevented their <i>in vivo</i> phosphopantetheinylation by AcpS, demonstrating D35N mutagenesis as a viable strategy to express apo-ACP in <i>E. coli</i>.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3986–3999"},"PeriodicalIF":3.0,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935914","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":"18-Crown-6-ether Utilizes Distinct Allosteric Interactions to Uncouple Transport by the Multidrug Efflux Pump EmrE","authors":"Merissa Brousseau,&nbsp;Tapasyatanu Dash,&nbsp;Michael J. Rourke,&nbsp;Vilius Kurauskas,&nbsp;Marco Tonelli,&nbsp;Kylie M. Hibbs,&nbsp;Andrew R. Buller,&nbsp;Michael T. Marty and Katherine A. Henzler-Wildman*,&nbsp;","doi":"10.1021/acs.biochem.5c00348","DOIUrl":"10.1021/acs.biochem.5c00348","url":null,"abstract":"<p >The recent discovery that the model multidrug efflux pump from <i>Escherichia coli</i>, EmrE, can perform multiple types of transport suggests that this may be a compelling target for therapeutic intervention. Initial studies have identified several small-molecule substrates capable of inducing transporter-dependent susceptibility rather than the well-known antibiotic resistance phenotype. However, many questions regarding the underlying mechanism and regulation of this transporter still remain. Prior studies identified lysine 22 as well as threonine 56 as important residues for regulating the formation of an occluded state critical to the prevention of an uncoupled leak in the WT transporter. Here, we use NMR chemical shift perturbations and <i>in vivo</i> EC₅₀ assays to confirm that 18-crown-6-ether binds at lysine 22, while liposomal leak assays verify that this substrate triggers uncoupled proton leak. In addition to characterizing the mechanism of action of another susceptibility substrate for EmrE, the characterization of K22 mutants herein solidifies the importance of this residue, as well as the nearby residue T56, in the allosteric regulation of the C-terminal tail. With a high degree of familial conservation in addition to a suggested role in transporter evolution, mechanistic insight into the transport regulation of EmrE may be broadly applicable across small multidrug-resistant efflux pumps.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3956–3970"},"PeriodicalIF":3.0,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12440375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935943","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 Insights into the Enhanced Antiviral Activity of Islatravir against Doravirine Resistance-Associated Substitution Mutations in HIV-1 Reverse Transcriptase.","authors":"Nikita Zalenski, Derek J Savoie, Amit Gaur, Neil A Patel, David J Suo, Turner W Seay, Daniel Betancourt, Zucai Suo","doi":"10.1021/acs.biochem.5c00397","DOIUrl":"10.1021/acs.biochem.5c00397","url":null,"abstract":"<p><p>Islatravir (ISL, EFdA) is a nucleoside analog that inhibits HIV-1 reverse transcriptase (RT) translocation during viral replication. Its high potency stems from unique structural features: a 4'-ethynyl group that interacts with the hydrophobic pocket (containing A114, Y115, F160, M184, and D185) in HIV-1 RT, hindering translocation, and a 3'-hydroxyl group that mimics natural nucleosides for efficient incorporation. Recent phase 3 clinical trials, combining ISL with Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, show that it is noninferior to existing treatments, offering a unique advantage due to their distinct resistance profiles. For instance, DOR-associated mutations, V106I/F227C, which confer >105-fold DOR resistance in clinics, unexpectedly boost ISL's potency by 2.3-fold in published cell-based resistance selection assays. In contrast, V106I alone does not affect Islatravir's potency, while F227C alone enhances it by 5.6-fold. To kinetically understand these findings, we used presteady-state kinetic assays to determine the kinetic parameters for EFdA 5'-triphosphate (EFdA-TP) and dATP incorporation. We found that the incorporation efficiency of EFdA-TP was 1.4-fold higher than that of dATP on an RNA template and 1.7-fold higher on a DNA template with the F227C mutant. However, this difference was only 1.1- to 1.3-fold higher with the F227C/V106I mutant. Our energy-minimized modeling revealed that these mutations remotely alter the hydrophobic 4'-ethynyl group-binding pocket's structure, surprisingly strengthening the pocket's binding interactions with EFdA-TP. Alongside this, the F227C mutation decreased dATP's binding affinity with both templates. Our data established a kinetic basis for the published cell-based resistance selection assay results, underscoring the significant potential of the ISL/DOR combination therapy in treating HIV-1 infected patients.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935645","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":"Differential Effects on In Vitro Tau Aggregation Due to 7, 11, and 14 Pseudophosphorylated Sites","authors":"Veena Prasad,&nbsp; and ,&nbsp;Truman C. Gamblin*,&nbsp;","doi":"10.1021/acs.biochem.5c00358","DOIUrl":"10.1021/acs.biochem.5c00358","url":null,"abstract":"<p >Neurofibrillary tangles are intracellular aggregates composed of the microtubule-associated protein tau. These insoluble aggregates are found in the brain of those affected by Alzheimer’s disease and other related tauopathies. Hyperphosphorylation of tau in disease has been hypothesized to cause tau to dissociate from microtubules and form amyloid-like oligomers and fibrils. Under normal conditions, there is 2–3 mol of phosphate per mole of tau; however, studies have found 2–3 times more phosphate per mole of tau in diseased conditions. The <i>in vitro</i> arachidonic acid induction of aggregation of different combinations of pseudophosphorylated sites up to 7 sites has previously been shown to result in differences in aggregation properties, characterized by increasing lengths of filaments with increasing numbers of pseudophosphorylation sites. Because several other sites of tau are also phosphorylated in disease, tau aggregation of protein variants with 11 and 14 sites mimicking hyperphosphorylation was compared to the 7 pseudophosphorylation sites previously studied using arachidonic acid and polyphosphate as fibrillization inducers. An increase in filament length, along with a decrease in the number of shorter filaments, was observed with increasing numbers of pseudophosphorylation sites regardless of the inducer employed. Variants displayed differential aggregation kinetics depending on the number of pseudophosphorylation sites and the inducer used. Although the rate of tubulin polymerization decreased as the number of pseudophosphorylation sites increased, microtubule stability was maintained across all pseudophosphorylated variants compared with unmodified wild-type tau. These results demonstrate that increasing levels of hyperphosphorylation can continue to have increased effects on tau aggregation and microtubule stabilization.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 18","pages":"3829–3840"},"PeriodicalIF":3.0,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935633","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":"Cysteine Mutagenesis of a Group II Intron-Encoded Protein Supports Splicing, Mobility, and Site-Specific Labeling.","authors":"Jasmine A Harper, Sarah A Starcovic, Neil Billington, Aaron R Robart","doi":"10.1021/acs.biochem.5c00382","DOIUrl":"10.1021/acs.biochem.5c00382","url":null,"abstract":"<p><p>Group II introns are self-splicing ribozymes that excise themselves from precursor RNA and integrate into new DNA locations through retromobility. Splicing is facilitated by an intron-encoded protein (IEP), a multidomain reverse transcriptase that enhances ribozyme activity and promotes formation of lariat intron-IEP ribonucleoprotein (RNP) complexes. In this study, we examined the role of conserved cysteine residues in the IEP of the group IIC intron <i>Ta.it.</i>I1 from the thermophile <i>Thermoanaerobacter italicus</i> by generating cysteine-to-methionine mutants. All variants retained near wild-type splicing efficiency, indicating that cysteine substitution does not impair maturase function. A mutation in the thumb domain significantly enhanced reverse transcription (RT) activity, whereas substitutions flanking the YADD catalytic motif led to reduced activity. Despite these variable RT effects, all mutants retained the ability to complete both steps of forward intron self-splicing and subsequently perform reverse splicing into DNA targets. Complete removal of native cysteines enabled site-specific fluorescent labeling of the IEP using maleimide-thiol chemistry without disrupting splicing or retromobility. Labeled IEPs retained activity and were successfully used to monitor RNA binding and RNP assembly under native conditions. These findings highlight the structural flexibility of IEP-intron interactions and demonstrate that site-specific IEP labeling enables real-time visualization of RNP assembly and dynamics.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144935621","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}