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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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22 recordsLinked to original sources

bioRxiv2026

Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein

The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S-glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo. Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly.

biochemistry↗

bioRxiv2026

Snapshots from the Catalytic Landscape of Chalcone Isomerase

Chalcone isomerase (CHI) catalyzes the cyclization of 3-ring scaffolds of flavonoids, a class of plant-based natural products important for nutrition and disease prevention. A persistent question has been whether the enzyme uses dynamics to facilitate conformational rearrangements of substrates within the active site. To help resolve this question, CHI was crystallized with phloretin, a flexible substrate analogue that cannot undergo cyclization. The crystal structure possesses eight protein molecules per asymmetric unit, revealing different active site conformations that accommodate different bound conformers of phloretin. Together, the structural snapshots depict a series of coordinated, dynamic chemical interactions that lower barriers to substrate rearrangements approaching bond formation. Differential scanning fluorimetry combined with mutational analysis and enzyme kinetics further confirm that phloretin binds to the enzyme active site and that it acts as a competitive inhibitor of CHI. Together these findings answer outstanding questions about the flexibility and dynamics of CHI catalysis, information that may be useful for future biosynthetic design and enzyme engineering goals. Overall, this work supports a catalytic model in which the CHI enzyme operates as a dynamic ensemble of structures necessary to facilitate catalytic substrate rearrangements.

biochemistry↗

bioRxiv2026

Structures of pUG-fold RNA bound to DNMT1 reveal a mechanism for RNA-mediated epigenetic regulation

Many chromatin-associated proteins have been found to bind RNA as a means of epigenetic regulation. Specifically, DNA methyltransferase 1 (DNMT1), which maintains cytosine methylation at CpG dinucleotides, is inhibited by RNA at transcribed DNA loci in cells. However, the mechanisms by which RNA binds DNMT1 and inhibits its activity remain unknown. Here, we determine a series of cryogenic electron microscopy (cryo-EM) structures of human DNMT1 bound to pUG-fold RNA, a non-canonical G-quadruplex previously observed to inhibit activity, revealing two distinct RNA-binding modes. The pUG-fold RNA binds the surface of DNMT1 in its autoinhibited conformation across a positively charged surface between the methyltransferase domain and the CXXC domain, and it binds directly in the active site of an open DNMT1 conformation. RNA binding is sterically incompatible with substrate DNA engagement in both states. Our 2.5 [A] structure captures the intricate network of hydrogen bonds and electrostatic interactions between amino acids in the methyltransferase domain and the tetrad layers of pUG-fold RNA. Metadynamics molecular dynamics simulations provide an orthogonal view of the conformational landscape of DNMT1, revealing the two distinct RNA-binding modes. Furthermore, our analysis of published DNMT1 RIP-seq and eCLIP-seq data confirms that DNMT1-interacting RNAs in cells exhibit a strong propensity to form non-canonical G-quadruplex RNA structures. Collectively, our study provides the first structural basis for pUG-fold RNA recognition by a protein and illustrates how cryo-EM and AI-based methods for protein and RNA structure prediction synergize to inform the mechanism of RNA-mediated regulation of DNMT1.

biochemistry↗

bioRxiv2026

Background proteome correction promotes confident identification of dynamic protein-protein interactions between different biological contexts

Affinity purification-mass spectrometry (AP-MS) enables the characterization of protein-protein interactions (PPIs), and the ease and sensitivity of such experiments has progressively increased. Beyond steady-state interactions of target proteins, a strong interest has emerged in monitoring how PPIs change upon significant biological perturbations, such as in disease contexts or small molecule modulation of the target protein. These perturbations likely not only induce PPI changes but can also lead to altered expression of proteins not of direct interest. Changes in protein abundance may alter which proteins adsorb to the affinity purification matrix, and due to the sensitivity of modern mass spectrometers, these differential ''background binders'' can masquerade as differential interactors. Contemporary approaches often do not account for differences in the background proteome, potentially inflating the number of false positives and negatives reported. Here, we provide technical considerations for the reliable annotation of dynamic PPIs, using the O-GlcNAc transferase (OGT) as a case study. We describe the installation of affinity epitope tags on endogenous OGT in mouse embryonic stem cells (mESCs), which we then apply for OGT interactor identification via AP-MS. We show that accurate representation of the bead background, which depends on the affinity matrix in use, is critical for elimination of false positive and false negative PPIs. This became even more pertinent as OGT PPI dynamics were measured under OGT catalytic inhibition via OSMI-4, which is known to perturb gene expression. The proteomes of OSMI-4-treated and control-treated mESCs differed, leading to distinct bead backgrounds in which the differential background proteins appeared as interaction gains or losses. These false positives were resolved by incorporating straightforward experimental controls through a practical statistical framework, allowing for a direct and confident comparison between treatment conditions. Incorporating these considerations into workflows investigating PPI dynamics will improve data fidelity and reproducibility.

biochemistry↗

bioRxiv2026

Gaussian accelerated Molecular Dynamics - Thermodynamic Integration (GaMD-TI): Improved alchemical free energy calculations with enhanced sampling

It is valuable to calculate alchemical free energy changes in drug discovery and development. Thermodynamics Integration (TI) has been widely used in computational chemistry for estimating free energy changes with alchemical transformations. However, TI based on usually short Molecular Dynamics (MD) simulations often suffers from insufficient conformational sampling. Here, we have integrated Gaussian accelerated MD and TI (GaMD-TI) to enhance the conformational sampling and improve accuracy of free energy calculations. GaMD-TI has been demonstrated in model systems of alchemical changes in the Valine dipeptide and mutation cycle of the Alanine <-> Valine <-> Isoleucine (AVI) residues. Simulations showed that when GaMD boost potentials followed near-Gaussian distribution, the free energy change could be reweighted accurately through generalized cumulant expansion to the second order. The total free energy change often exhibited faster convergence using Selective GaMD (SGaMD) than using conventional MD (cMD). Accuracy of the free energy estimates from SGaMD-TI simulations was similar to or higher than those from cMD-TI simulations, although the differences were subtle for these small model systems. Meanwhile, dihedral angles in the model systems underwent significantly more frequent conformational transitions in SGaMD than in cMD, indicating improved sampling. Future studies are planned on larger systems with more complicated alchemical changes, such as ligand binding to proteins/nucleic acids and mutations at biomolecular binding interfaces. GaMD-TI should be broadly applicable to alchemical free energy calculations and therapeutic design.

biochemistry↗

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Structural basis for tetraspanin-dependent surface export and adhesive function of integrin α3β1

Integrin 3{beta}1 (ITG3{beta}1) is a member of an integrin subfamily that binds to laminin proteins and promotes attachment of epithelial cells to the basement membrane. ITG3{beta}1 forms a complex with the tetraspanin CD151, and loss-of-function mutations in both ITG3 and CD151 cause epidermolysis bullosa, a severe skin blistering disease resulting from a defect in basement membrane attachment. Here, we report the cryoEM structure of an ITG3{beta}1 complex with CD151 and show that mutation of CD151 at the binding interface disrupts complex formation in cells. Strikingly, CRISPR-mediated knockout of CD151 leads to a variably penetrant ITG3{beta}1 surface export defect that is restored by re-expression of wild-type but not interface-mutated CD151. Together, these studies define the molecular basis for binding of CD151 to ITG3{beta}1, and show that CD151 promotes ITG3{beta}1 surface export, providing a biochemical explanation for the CD151 loss-of-function phenotype in epidermolysis bullosa.

biochemistry↗

bioRxiv2026

Cryo-EM structures reveal the mechanism of phosphatidylserine remodeling by membrane-bound glycerophospholipid O-acyltransferase 1

Lands cycle remodeling of glycerophospholipid acyl chains is crucial for cells to maintain appropriate membrane composition. Glycerophospholipids are cleaved at the glycerol sn2-position by phospholipase A. The lysophospholipids are reacylated by enzymes of the membrane-bound O-acyltransferase (MBOAT) family to incorporate specific fatty-acyl chains to adjust membrane properties. How MBOAT enzymes recognize specific acyl-CoA donors, select lysophospholipid acceptors, and release products is unclear. Phosphatidylserine (PS), a critical anionic phospholipid, controls membrane surface charge, signaling-protein recruitment, and cell-death-associated membrane recognition, and PS acyl-chain remodeling is linked to ferroptosis resistance. Here, we showed that MBOAT1 preferentially generates monounsaturated fatty acid-containing PS from lyso-PS. High-resolution cryo-electron microscopy structures of human MBOAT1 captured distinct binding poses of the fatty acyl donor, lyso-PS acceptor, and PS product. With lipidomics, enzymology and molecular dynamics simulations, these structures reveal the mechanism and pathway of MBOAT1-dependent PS remodeling.

biochemistry↗

bioRxiv2026

Temozolomide Induces Aberrant RNA Alkylation and Widespread Translational Repression

Temozolomide (TMZ) is a frontline alkylating chemotherapy, yet its direct impact on RNA modification and global translation dynamics remains poorly understood. Here, we demonstrate that TMZ induces pervasive RNA alkylation causing severe translational impairment. TMZ directly deposits aberrant methyl groups onto single-stranded mRNA in vitro, creating physical lesions that lower translational efficiency. In glioblastoma cells, acute TMZ exposure triggers a rapid, widespread accumulation of m7G on cellular RNAs, leading to the significant attenuation of global protein synthesis. Nanopore direct RNA sequencing identified distinct guanine-specific error signatures and sequence context preferences associated with TMZ-induced damage. Using a quantitative yeast spike-in ribosome profiling strategy, we mapped this translational repression at transcript-level, revealing a global downregulation of translational efficiency. This widespread repression disproportionately targets highly interconnected networks essential for cellular proliferation, specifically chromosome organization. We show that the severity of this translational repression is driven by a transcript's coding guanine density, stability and translation initiation speed. Together, our findings suggest that TMZ-induced alkylation targets stable, highly translated, guanine-rich transcripts. This establishes aberrant RNA methylation and subsequent translational arrest as a potential mechanism of temozolomide cytotoxicity.

biochemistry↗

bioRxiv2026

Sequential Molecular Interactions Shape Aβ42 Aggregation, Propagation, and Toxicity

Protein aggregation is a context-dependent process in which the molecular environment can influence the properties of the resulting assemblies. In biological systems, these interactions can occur sequentially, as aggregates formed in one cellular or tissue context may encounter different molecular partners and act as seeds in subsequent aggregation events. Here, we used sequential seeding as a controlled experimental model of this temporal and contextual complexity to investigate how prion-like sequences from the gut microbiome modulate amyloid-{beta} aggregation across successive aggregation cycles. Combining kinetic, biophysical, conformational, and toxicity analyses, we show that early interactions with exogenous peptides modify the properties of first-generation A{beta}40- and A{beta}42-derived seeds, resulting in propagated A{beta}42 assemblies with distinct molecular and functional properties. These findings support an Interaction History model in which exogenous sequences bias the emergence of aggregate populations whose properties and subsequent propagation depend on the molecular contexts experienced during earlier aggregation events. Overall, our results present A{beta} aggregation as a history-dependent process and suggest that single-step assays may fail to capture aggregate diversity that emerges across successive aggregation cycles.

biochemistry↗

bioRxiv2026

Mind the gap between functional groups and surface of magnetic nanoparticles for highly specific magnetic-based protein assays in biological medium

Magnetic readout-based assays are compatible with unprocessed biological samples as unbound background molecules do not interfere with magnetic signal. Yet, a true challenge is their poor specificity and susceptibility of magnetic nanoparticles (MNPs) to clusters in complex biological media, hampering their true advancement. Here, we demonstrate that the spatial organization of functional groups at the external periphery of custom magnetic nanoparticles by harnessing ultra-dense double-stranded DNA results in an efficient antibody conjugation with good accessibility toward antigen. By labeling our MNPs with anti-S protein neutralizing IgG antibody, we showcase the detection of S1 subunit of SARS-CoV-2 Spike protein in a wash-free fashion in less than five minutes in nM regime using magnetic particle spectrometer. By mixing our IgG-labelled MNPs with DMEM cell culture (10-20% FBS serum), we sense the S1 proteins in a one-pot fashion with high specificity. Our results show that by having the ultra-dense dsDNA shell on MNPs, the entropic cost of an irreversible protein binding to particle surface is high, thus allowing the formation of dynamic protein corona on the DNA shell that can be replaced with S1 protein with high affinity. When the azide moieties are placed at the close proximity of MNPs by using non-functional dsDNA, antibody conjugation becomes inefficient, to a level not sufficient for S1 protein detection. Our study highlights the importance of spatial organization of functional moieties on the nanoscale on magnetic nanoparticles for highly specific assays in biologically complex media.

biochemistry↗

bioRxiv2026

Structure-inspired design of Nsp8-based protein inhibitors to suppress SARS-CoV-2 replication

SARS-CoV-2 relies on a conserved RNA-dependent RNA polymerase (RdRp) complex composed of nsp12 and its cofactors nsp7 and nsp8 to replicate its RNA genome. Whereas most antiviral strategies target viral enzymes or surface proteins directly, an alternative approach is to disrupt the assembly or function of an essential viral molecular machine using a defective component derived from the pathogen itself. Here, guided by structural analyses of the nsp12-nsp7-nsp8 replication complex, we designed truncated nsp8 proteins that retain the ability to associate with nsp12 but are defective in engaging RNA. Using a purified nsp12-nsp7-nsp8 system capable of RNA primer extension, we show that selected truncated nsp8 variants inhibit polymerase activity when introduced into an otherwise functional complex. These results are consistent with a competitive mechanism in which the defective nsp8 variants associate with nsp12 and interfere with incorporation or function of wild-type nsp8, thereby compromising formation of a productive replication complex. To further explore this strategy, we used structure-guided in silico analysis of the nsp8-nsp12 interface to identify interaction hotspots and screened corresponding single-amino-acid substitutions. Several variants exhibited enhanced inhibitory activity in the reconstituted polymerase assay. Together, these findings establish a proof-of-concept strategy in which a structurally engineered, pathogen-derived protein can act as a dominant-negative inhibitor of an essential viral replication machinery. This approach provides a framework for developing protein- or peptide-based inhibitors that target conserved protein-protein interactions within viral replication complexes.

biochemistry↗

bioRxiv2026

Scaffold Affinity Tunes Biomolecular Condensate Function

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

biochemistry↗

bioRxiv2026

Structural mechanism defining product specificity in glycoside hydrolase family 66 cycloisomaltotetraose glucanotransferase

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

biochemistry↗

bioRxiv2026

A family-wide atlas of human connexin docking compatibility

Gap junction (GJ) channels mediate direct intercellular communication by allowing the exchange of ions, metabolites, and signaling molecules between neighboring cells. Humans express 21 connexin (Cx) isoforms that can assemble into homotypic or heterotypic channels, creating a large potential interaction landscape that shapes tissue-specific communication networks. However, the rules governing which connexin isoforms can compatibly dock remain incompletely defined. Extracellular loop 2 (EL2) sequence features have been implicated in docking specificity and used to classify connexins into two canonical compatibility groups, K-N and H, but these assignments remain largely predictive. Most potential heterotypic connexin pairings have never been experimentally tested. This incomplete interaction map limits our ability to predict which connexin combinations can assemble, how isoform co-expression shapes intercellular communication, and how these relationships are altered or exploited in disease and engineered systems. Here, we used the FETCH (Flow Enabled Tracking of Connexosomes in HEK Cells) assay to evaluate docking compatibility across the complete human connexin family. To support family-wide compatibility mapping, we used literature-supported heterotypic interactions to define a data-driven FETCH score threshold for high-confidence interaction compatibility. Homotypic FETCH measurements varied substantially across the 21 connexin isoforms, with 15 producing mean scores above the empirical threshold. We then extended FETCH analysis to all 210 pairwise heterotypic isoform combinations. The resulting interaction landscape largely recapitulated expected motif-class relationships, including enrichment within the two canonical compatibility groups, but also identified neighboring-group interactions and unexpected cross-group pairings that represented clear exceptions to class-based predictions. Consistent with these findings, pairwise EL2 motif similarity was only modestly associated with threshold-based interaction classification, indicating that EL2 similarity alone was insufficient to predict compatibility outcomes. Together, these findings suggest that motif class provides a broad organizing framework for connexin compatibility, but that pairwise docking specificity also depends on yet-unresolved isoform-specific determinants that produce neighboring-group relationships and clear cross-group exceptions. Notably, Cx46, a lens Cx also associated with melanoma and breast cancers, emerged as a broadly permissive isoform capable of interacting with partners from both major compatibility groups and more than half of the connexin family. Together, these findings establish the first family-wide experimental atlas of human connexin docking compatibility, defining canonical interactions, previously unrecognized pairings, and exceptions to established compatibility rules. This atlas provides a foundation for defining the molecular determinants of connexin specificity, understanding how isoform diversity shapes intercellular communication, and designing gap junction channels with controlled docking behavior.

biochemistry↗

bioRxiv2026

IKK2/β mediated phosphorylation of transcription factor Ets2 at site(s) distal to DNA binding domain negatively modulates its DNA binding activity

Transcription factor Ets2 coalesce with the NF-{kappa}B pathway to regulate gene expression in specific signaling contexts. IKK2/{beta}-mediated phosphorylation events critically regulate the NF-{kappa}B pathway. However, any link between Ets2 and IKK2 remains elusive. Here we report Ets2 as a direct substrate of IKK2. In-vitro kinase assays using deletion constructs, high resolution MS-MS and site directed mutagenesis identified S295 as a prominent phosphorylation site distal to the DNA binding domain, substitution of which to phosphor-mimetic Glutamate triggers further phosphorylation of Ets2. MD simulations clearly indicate conformational constriction of the otherwise disordered N-terminal region and inhibition of DNA binding activity upon phosphorylation, which was further confirmed by Electrophoretic mobility shift assays. Our results uncover a phosphoregulatory connection between Ets2 and IKK2.

biochemistry↗

bioRxiv2026

Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. For the first time, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We next introduced specific mismatches to the gRNA. Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage dynamics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they redirected editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

biochemistry↗

bioRxiv2026

Cryo-EM Structure of a Triazole alpha-Conotoxin GI Mimetic Bound to the Muscle-Type Nicotinic Acetylcholine Receptor

Disulfide-rich peptides possess exceptional potency and selectivity but are often limited by the instability and synthetic challenges associated with native disulfide bonds. Here, we report the design, synthesis, pharmacological evaluation, and structural characterisation of triazole-based peptidomimetics of the -GI conotoxin, a selective antagonist of the muscle-type nicotinic acetylcholine receptor (nAChR). A series of 1,4- and 1,5-disubstituted triazole analogues were prepared entirely on resin using CuAAC and RuAAC chemistry to replace the native Cys3/13 disulfide bridge. Functional evaluation against human muscle nAChRs revealed that 1,5-triazole analogues retained low-nanomolar potency, with the lead mimetic exhibiting activity comparable to native -GI. Cryo-electron microscopy of the lead compound bound to the muscle-type nAChR provided the first structure of a disulfide-isostere peptidomimetic in complex with a membrane receptor. The structure demonstrates that the 1,5-triazole reproduces the native peptide fold with high fidelity while contributing receptor-facing interactions not available to the native disulfide bridge. Molecular dynamics simulations further revealed conserved hydration networks and similar conformational sampling between the native peptide and lead mimetic. Together, these findings establish triazoles as effective disulfide surrogates and provide a structural framework for the rational design of stabilised conotoxin therapeutics.

biochemistry↗