Biochemistry Biochemistry最新文献

Theaggregation of transthyretin (TTR) is a hallmark of ATTR amyloidosis.Accumulating evidence suggests that metal ions, particularly Zn²⁺, promote TTR aggregation; however, the molecular mechanismunderlying Zn²⁺-induced misfolding remains poorly understood.Here, we present a comprehensive biophysical characterization of Zn²⁺-induced misfolding of wild-type transthyretin (TTRwt) underphysiologically relevant conditions. Two-dimensional ¹H/¹⁵N HSQC NMR analyses reveal that Zn²⁺ binding inducesmillisecond-time scale conformational exchange that destabilizes thenative tetramer. These structural and dynamic perturbations promotethe formation of small, toxic oligomeric species capable of self-propagationthrough the seeding of native TTR. Comparative structural analysesusing solid-state NMR also show that Zn²⁺-induced oligomersadopt broadly native-like secondary structures, but exhibit distinctnon-native molecular conformations. The early stages of misfoldingwere further investigated using ¹⁹F NMR of TTRwt containingfluorinated tryptophan residues. Notably, ¹⁹F NMR not onlyreports on Zn²⁺ binding but also enables direct detectionof a transient monomeric intermediate generated upon tetramer dissociation,a key species in the amyloidogenic cascade. Together, these findingsdefine the structural and dynamic basis of Zn²⁺-mediatedTTR misfolding and establish combined solution and solid-state NMRapproaches as powerful tools for probing metal-induced misfoldinglandscapes in TTR and related amyloidogenic proteins.

Alkylation at N7 of guanine leads to formation of abasic(AP) sitesin DNA. These equilibrate between α and β anomers andform conjugates with 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione (Pixantrone, PIX). NMR revealed that theseconjugates formed as regioisomeric Schiff base products R_a and R_b arising from thenonequivalent PIX C6 and C9 2-aminoethyl side chains. In dsDNA formationof R_a was favored. In ssDNA, both productsformed equally. Structures of reduced R_a and R_b conjugates from molecular dynamicscalculations restrained by ¹H NOESY data in 5′-d(C¹A²G³A⁴C⁵T⁶X⁷A⁸G⁹A¹⁰C¹¹T¹²)-3′:5′-d(A¹³G¹⁴T¹⁵C¹⁶T¹⁷C¹⁸A¹⁹G²⁰T²¹C²²T²³G²⁴)-3′, X = reducedPIX-AP site conjugate, showed that both regioisomers intercalatedon the 3′ side of base pair T⁶/A¹⁹, displacingC¹⁸ into the major groove, with R_a less intercalated than R_b. Thepreferential formation of R_a vs R_b in dsDNA and deeper intercalation of R_b may be explained by the differential abilityof the asymmetric PIX N2 nitrogen in R_a vs R_b to hydrogen bond with the exocyclic N⁴-amine of the cytosine displaced into the majorgroove. MD simulations in dsDNA with R_a and R_b reduced PIX AP site conjugatesrevealed that for both, the isoquinoline first reoriented within theminor groove. The complementary cytosine rotated toward the majorgroove, allowing intercalation of the R_a and R_b conjugates. Binding energy analysesyielded values of −108.1 (±9.7) kcal/mol and −102.2(±8.3) kcal/mol for the intercalated R_a and R_b conjugates, respectively. A crystalstructure in the Dickerson-Drew Dodecamer at 2.5 Å resolutionrevealed looped-out PIX stacking with PIX from a neighboring duplex.

Ionotropic glutamate receptors (iGluRs) mediate rapidsignals betweenneurons by binding extracellular neurotransmitters and conductingionic current across the cell membrane. Animal iGluRs can be dividedinto clades of phylogenetically related receptors, only some of whichare present in mammals. Despite their name, many receptors are selectivelyactivated by ligands other than glutamate, and despite high-resolutionstructural data on most mammalian iGluRs, the molecular basis forligand selectivity in most clades is not clear. We questioned whetherclosely related iGluRs within phylogenetic clades share similar ligandselectivity profiles by testing several receptors from bilaterians,cnidarians, a placozoan, and a ctenophore, and by probing the molecularbasis of ligand selectivity in iGluRs from different clades with site-directedmutagenesis. We identified glutamate-, glycine-, and GABA-gated channelsfrom both the AKDF clade and the Epsilon clade, and a selectivelyaspartate-gated channel from the Epsilon clade. We show that in somecases, even very closely related channels from within the same cladeand from the same animal species have diverged functionally, leadingto channels gated by different agonists. Although we find specialroles in ligand selectivity for glycine, threonine, and leucine residuesin the “αF-GST” and “β10/αI-LE”motifs (mammalian AMPA receptor nomenclature), our results in diverseiGluRs warn that the effects of mutations, and thus the roles of theseresidues, are not always easy to predict.

Lassa virus (LASV),a member of the arenavirus family, utilizeslow-pH-dependent membrane fusion to enter host cells and elicit infection.LASV is a class I fusion virus, and thus its fusion protein, GP2,is functionally similar to other class I fusion proteins in that itfollows the six-helix bundle fusion mechanism. LASV fusion is atypicalin that it requires another viral protein, stable signal peptide (SSP),that does not undergo fusion itself but is required for the fusionprocess. However, the exact function of SSP in fusion is unknown.We aimed to understand the role of SSP in the fusion process by examiningthe structural changes that occur within SSP during the fusion process.We found that SSP undergoes two distinct structural changes; the firstat pH 6 that is not associated with fusion, the second at below pH5 that is associated with fusion activity. Furthermore, we found amutation to a key residue of SSP, K33A, does not undergo these twostructural changes; in fact, K33A is seen to undergo a different conformationalchange at pH 6. We believe this distinct conformation change is dueto the loss of favorable interactions between the lysine at the endof the transmembrane helix of SSP. This disruption of favorable interactionthen causes downstream effects to the SSP structure in the event oflowering pH. The difference in conformational changes between K33Aand wildtype (WT) SSP is likely why K33A abrogates the fusion activityof GP2. We conclude that the conformational changes undergone by SSPare crucial for LASV fusion activity.

Functionalizing DNAnanostructures for stimuli-responsivereconfigurationhas been a key area of development in DNA nanotechnology. Here, wedemonstrate programmable control over the structural integrity ofa tetrahedral DNA nanostructure using an external DNA stimulus thatis modulated through an intermediate CRISPR-Cas12a reaction. We designedtoehold-containing DNA tetrahedra that can be disassembled by a DNAor RNA invading strand. Activation of the Cas12a enzyme by double-strandedDNA provides precise control over the disassembly of the DNA tetrahedraby digesting the DNA invading strand but not the RNA, enabling highlyresponsive and programmable DNA architectures. Further, targeted activationof Cas12a by conserved genomic regions of three bacterial pathogens,namely Listeria monocytogenes, Campylobacter jejuni, and Shiga toxin-producing Escherichia coli, provides biologically relevant responsiveness and demonstratesthe platform’s potential for future applications. Our resultsshow that the DNA tetrahedra are observed in the presence of the genomictarget but not observed in its absence due to disassembly, demonstratinga highly programmable and responsive molecular level architecture.

UFMylation is an important biological process where proteinsarepost-translationally modified via the covalent attachment of the ubiquitin-likemodifier (UBL), ubiquitin-fold modifier-1 (UFM1). UFMylation is analogousto ubiquitination, occurring via the transfer of UFM1 across E1-,E2-, and E3-like enzymes, represented by ubiquitin-like modifier-activatingenzyme 5 (UBA5), ubiquitin-fold modifier conjugating enzyme 1 (UFC1),and UFM1-specific ligase 1 (UFL1), respectively. Recent work has shownthat dysregulation of UFMylation is associated with several diseases,sparking interest in characterizing its enzymes as potential drugtargets. To date, only inhibitors targeting UBA5 have been identified,but no such ligands exist for UFC1. In this study, we present thestructure of UFC1 in complex with the sulfonic acid CAPS, which revealeda novel ligand-binding pocket in UFC1. Using biophysical assays, coupledwith X-ray crystallographic studies of UFC1 variants, we show thatbinding of CAPS to UFC1 appears pH-dependent and is enhanced by Tyr42.Further, our TSA data show that other sulfa- and sulfonate-based compoundsinduce dose-dependent destabilization of UFC1, consistent with weakbut direct interactions with the enzyme. Lastly, using a UFMylationassay, we show that CAPS, along with Tyr42, may have a limited influenceon UFM1 transfer to UFC1 and, consequently, downstream UFMylationof protein substrates. Nevertheless, our data indicate that the CAPS-bindingpocket may serve as a design scaffold for the development of UFC1modulators. With UFC1 emerging as a drug target, our study providesa possible avenue for the design and development of novel UFC1-specificmodulators with therapeutic potential.

Cross-linking mass spectrometry (XL-MS) with photoactivatablereagents has been increasingly applied to probe protein conformational dynamics;however, its sensitivity to shifts in conformational equilibrium involving transient, sparsely populated docked states within multidomainproteins remains largely unexplored. Mammalian nitric oxide synthase(NOS), a family of modular redox enzymes, relies on large-amplitudeFMN domain swinging motions and local, dynamic interdomain FMN–hemedocking to function. This makes it a stringent test case for evaluatinghow sensitive photoactivated XL-MS is to shifts in the docking equilbrium.In our previous work, quantitative XL-MS using the primarily lysine-reactivehomobifunctional cross-linker DSBU revealed E546N mutation-induced decreases in specific intersubunit FMN–heme cross-links inan oxygenase/FMN construct of human inducible NOS (iNOS). Here, weextended these XL-MS studies to sulfo-SDA, a heterobifunctional, photoactivatablecross-linker with ultrafast reactivity and broad residue specificity.SDA captured interdomain FMN–heme cross-links at the dockinginterfaces but, unlike DSBU, did not reveal statistically robust changesin the abundances of these cross-links between wild-type and E546Nmutant. SDA produced a higher proportion of dead-end labeling productsfor iNOS oxyFMN proteins, particularly the E546N mutant, than forthe conformationally stable protein BSA. Moreover, the SDA cross-linkdistance restraints were largely satisfied by a minimal subset of docked states with distinct FMN domain orientations, whereas DSBUcross-links require a combination of docked and undocked conformationsfor optimal restraint satisfaction. These findings reflect the shorteffective distance range and near-instantaneous reactivity of SDA,which restrict FMN–heme cross-link formation to a narrow subsetof transient docking states. Taken together, these results underscorethe importance of selecting appropriate cross-linking reagents andemploying complementary cross-linkers to define the conformationallandscapes of dynamic multidomain proteins.

Protein–DNA complexes are stabilized by variousinteractionsthrough an interaction network between protein and DNA. Any changein the system─whether through mutations in protein/DNA, externalfactors, or protein conformational transitions─can alter theinteraction network, affecting structural and functional aspects.Employing all-atom classical molecular dynamics, we investigated howthe interaction network in ZTA–DNA is rewired, while key arginineresidues in ZTA are mutated to oppositely charged glutamic acids.Using the MM/PBSA technique, we computed per-residue binding energiesand correlated them with structural features. A detailed mechanisticstudy shows that mutations in key arginine residues form new interactionseither around the mutation site and/or in the other ZTA monomer. Throughload-sharing system attempts to counterbalance the interaction load,leading to reorganization of the existing interaction network. Fromsingle- to double-site mutations, the complex partially maintainsits structural stability through additional interactions formed bylysine, particularly K178, while multisite mutations cannot sustainits structural stability, leading to system destabilization. Despiteinherent structural symmetry in ZTA, an asymmetric monomer contributionis observed upon mutation. The binding affinity of the ZTA–DNAcomplex shows a good correlation with structural and interaction features,following the trend A > E ≈ D > C ≫ B. Overall,ourrigorous mechanistic study provides deeper insights into the interactionnetwork reorganization mechanism in the ZTA–DNA system. SinceZTA is a key factor in the Epstein–Barr virus (EBV), this studywill be central to understanding DNA recognition and developing drugtherapeutics targeting viral transcription factors in EBV.

Posttranslationallysine acetylation and methylationare generallyconsidered mutually exclusive modifications of the ε-amino group.The recent discovery of N-acetyl-N-methyl lysine (KAcMe) on histone H4 showed that both marks can coexiston the same lysine side chain, raising the question of whether thisdual modification is enzymatically reversible. Here, we synthesizedmodel peptides containing N-acyl lysine or N-acyl-N-methyl lysine residues and testedtheir processing by Zn²⁺-dependent HDACs and NAD⁺-dependent sirtuins. While the corresponding unmethylated substrateswere deacylated by the expected enzymes, KAcMe-containing peptideswere not detectably deacetylated by either enzyme family. N-methylation also blocked HDAC8/11-mediated demyristoylationand SIRT5-mediated desuccinylation, whereas SIRT2 and SIRT6 retainedweak demyristoylase activity toward N-myristoyl-N-methyl lysine. X-ray crystal structures of sirtuin–peptidecomplexes, supported by inhibition studies and molecular docking,reveal that N-methylation disrupts productive substratepositioning and misaligns the scissile amide bond for catalysis. Thesefindings suggest that KAcMe is resistant to canonical lysine deacetylasesand may represent a persistent chromatin-associated modification.

Protein misfolding and aggregation in neuronal cellsare key molecularevents underlying several fatal neurodegenerative disorders. Effectivetherapeutic intervention requires residue-resolved structural informationon the early aggregation intermediates that precede amyloid formation.However, these transient and heterogeneous species remain poorly characterizedbecause of their dynamic nature and the limitations of conventionalstructural approaches. Here, we employ a multisite fluorescence approachto obtain residue-specific insights into the misfolding and aggregationpathway of the nucleic acid-binding domain of TDP-43. Using a libraryof single-tryptophan–single-cysteine mutants combined withsite-selective labeling, we systematically probe intramolecular distances,local solvation, conformational rigidity, and structural fluctuationsacross native, molten globule, oligomeric, and amyloid states. Theresults reveal that the early amyloidogenic molten globule retainsnative-like secondary structure but undergoes heterogeneous structuralreorganization, including simultaneous local expansion and compaction,as revealed by multisite Förster resonance energy transfer.Residue-specific analyses also identify early unfolding sites andregions that progressively become incorporated into the amyloid core.These findings provide a residue-resolved view of the structural transitionsthat link native conformations to aggregation-prone states and establisha framework for understanding early events in TDP-43 misfolding.