Non-canonical Motif Research Articles

Computational Identification and Illustrative Standard for Representation of Unimolecular G-Quadruplex Secondary Structures (CIIS-GQ).

G-quadruplexes refer to a large group of nucleic acid-based structures. In recent years, they have been attracting attention due to their biological roles in the telomeres and promoter regions. These structures show wide diversity in topology, however, development of methods for structural classification of G-quadruplexes has been evaded for a long time. There has been a limited number of studies aiming to bring forth a secondary structure classification method. The situation was even more complex than imagined, since the discovery of bulged and mismatched G-quadruplexes while most of the available tools fail to distinguish these non-canonical G-quadruplex motifs. Moreover, the interpretation of their analysis output still requires expert knowledge. In this study, we propose a new method for identification of unimolecular G-Quadruplexes and classification by secondary structures based on three-dimensional structural data. Briefly, coordinates of guanines are processed to identify tetrads, loops and bulges. Then, we present the secondary structure in the form of a depiction which shows the loop types, bulges, and guanines that participate in each tetrad. Moreover, CIIS-GQ identifies non-guanine nucleotides that joins the G-tetrads and forms multiplets. Finally, the results of our study are compared with DSSR and ElTetrado classification methods, and the advantages of the proposed depiction method for representing secondary structures were discussed. The source code of the method can be accessed via https://github.com/TugayDirek/CIIS-GQ .

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity invitro.

The First FISH-Confirmed Non-Canonical Telomeric Motif in Heteroptera: Cimex lectularius Linnaeus, 1758 and C. hemipterus (Fabricius, 1803) (Hemiptera, Cimicidae) Have a 10 bp Motif (TTAGGGATGG)n.

Fluorescence in situ hybridization (FISH) with two different probes, the canonical insect telomeric sequence (TTAGG)n and the sequence (TTAGGGATGG)n, was performed on meiotic chromosomes of two members of the true bug family Cimicidae (Cimicomorpha), the common bed bug Cimex lectularius Linnaeus, 1758 and the tropical bed bug C. hemipterus (Fabricius, 1803), whose telomeric motifs were not known. In both species, there were no hybridization signals with the first probe, but strong signals at chromosomal ends were observed with the second probe, indicating the presence of a telomeric motif (TTAGGGATGG)n. This study represents the first FISH confirmation of the presence of a non-canonical telomeric motif not only for the infraorder Cimicomorpha but also for the suborder Heteroptera (Hemiptera) as a whole. The present finding is of key significance for unraveling the evolutionary shifts in the telomeric sequences in this suborder.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Exploring mechanisms of mupirocin resistance and hyper-resistance.

Mupirocin is a broad-spectrum antibiotic that acts predominantly against Gram-positive bacteria. It is produced by Pseudomonas fluorescens NCIMB 10586 and has been clinically used to treat primary and secondary skin infections and to eradicate nasal colonisation of methicillin-resistant Staphylococcus aureus strains. Mupirocin inhibits protein synthesis by blocking the active site of isoleucyl-tRNA synthetase (IleRS), which prevents the enzyme from binding isoleucine and ATP for Ile-tRNAIle synthesis. Two types of IleRS are found in bacteria - while IleRS1 is susceptible to mupirocin inhibition, IleRS2 provides resistance to cells. These two types belong to distinct evolutionary clades which likely emerged from an early gene duplication in bacteria. Resistance in IleRS2 is based on the loss of interactions that govern mupirocin binding to IleRS1, such as hydrogen bonding to the carboxylate moiety of mupirocin. IleRS2 enzymes with Ki in the millimolar range have recently been discovered. These hyper-resistant IleRS2 variants surprisingly have a non-canonical version of the catalytic motif, which serves as a signature motif of class I aminoacyl-tRNA synthetases to which IleRS belongs. The non-canonical motif, in which the 1st and 3rd positions are swapped, is key for hyper-resistance and can be accommodated without abolishing enzyme activity in IleRS2 but not in IleRS1. Clinical use of mupirocin led to the emergence of resistance in S. aureus. Low-level resistance arises by mutations of the housekeeping IleRS1, while high-level resistance develops by the acquisition of the resistant IleRS2 on a plasmid. There is no evidence that hyper-resistant variants have been found in clinical isolates.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification.

Sitting on the interface between biologics and small molecules, peptides represent an emerging class of therapeutics. Numerous techniques have been developed in the past 30 years to take advantage of biological methods to generate and screen peptide libraries for the identification of therapeutic compounds, with phage display being one of the most accessible techniques. Although traditional phage display can generate billions of peptides simultaneously, it is limited to expression of canonical amino acids. Recently, several groups have successfully undergone efforts to apply genetic code expansion to introduce noncanonical amino acids (ncAAs) with novel reactivities and chemistries into phage-displayed peptide libraries. In addition to biological methods, several different chemical approaches have also been used to install noncanonical motifs into phage libraries. This review focuses on these recent advances that have taken advantage of both biological and chemical means for diversification of phage libraries with ncAAs.

Analysis of REST binding sites with canonical and non-canonical motifs in human cell lines

BackgroundRepressor element 1 (RE1) silencing transcription factor (REST) is a transcriptional repressor abundantly expressed in aging human brains. It is known to regulate genes associated with oxidative stress, inflammation, and neurological disorders by binding to a canonical form of sequence motif and its non-canonical variations. Although analysis of genomic sequence motifs is crucial to understand transcriptional regulation by transcription factors (TFs), a comprehensive characterization of various forms of RE1 motifs in human cell lines has not been performed.ResultsHere, we analyzed 23 ENCODE REST ChIP-seq datasets from diverse human cell lines and identified a non-redundant set of 68,975 loci with ChIP-seq peaks. Our systematic characterization of these binding sites revealed that the canonical form of REST binding motif was found primarily in ChIP-seq peaks shared across multiple cell lines, while non-canonical forms of motifs were identified in both cell-line-specific binding sites and those shared across cell lines. Remarkably, we observed a notable prevalence of non-canonical motifs that corresponded to half segments of the canonical motif. Furthermore, our analysis unveiled the presence of cell-line-specific REST binding patterns, as evidenced by the clustering of ChIP-seq experiments according to their respective cell lines. This observation underscores the cell-line specificity of REST binding at certain genomic loci, implying intricate cell-line-specific regulatory mechanisms.ConclusionsOverall, our study provides a comprehensive characterization of REST binding motifs in human cell lines and genome-wide RE1 motif profiles. These findings contribute to a deeper understanding of REST-mediated transcriptional regulation and highlight the importance of considering cell-line-specific effects in future investigations.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

DNA minicircles capable of forming a variety of non-canonical structural motifs.

Although more than 10% of the human genome has the potential to fold into non-B DNA, the formation of non-canonical structural motifs as part of long dsDNA chains are usually considered as unfavorable from a thermodynamic point of view. However, recent experiments have confirmed that non-canonical motifs do exist and are non-randomly distributed in genomic DNA. This distribution is highly dependent not only on the DNA sequence but also on various other factors such as environmental conditions, DNA topology and the expression of specific cellular factors in different cell types. In this study, we describe a new strategy used in the preparation of DNA minicircles containing different non-canonical motifs which arise as a result of imperfect base pairing between complementary strands. The approach exploits the fact that imperfections in the pairing of complementary strands thermodynamically weaken the dsDNA structure at the expense of enhancing the formation of non-canonical motifs. In this study, a completely different concept of stable integration of a non-canonical motif into dsDNA is presented. Our approach allows the integration of various types of non-canonical motifs into the dsDNA structure such as hairpin, cruciform, G-quadruplex and i-motif forms but also combinations of these forms. Small DNA minicircles have recently become the subject of considerable interest in both fundamental research and in terms of their potential therapeutic applications.

Sequence composition changes in short tandem repeats: heterogeneity, detection, mechanisms and clinical implications.

Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms.

Sequence and structural insights of monoleucine-based sorting motifs contained within the cytoplasmic domains of basolateral proteins.

Delivery to the correct membrane domain in polarized epithelial cells is a critical regulatory mechanism for transmembrane proteins. The trafficking of these proteins is directed by short amino acid sequences known as sorting motifs. In six basolaterally-localized proteins lacking the canonical tyrosine- and dileucine-based basolateral sorting motifs, a monoleucine-based sorting motif has been identified. This review will discuss these proteins with an identified monoleucine-based sorting motif, their conserved structural features, as well as the future directions of study for this non-canonical basolateral sorting motif.

Concurrent prediction of RNA secondary structures with pseudoknots and local 3D motifs in an integer programming framework.

The prediction of RNA structure canonical base pairs from a single sequence, especially pseudoknotted ones, remains challenging in a thermodynamic models that approximates the energy of the local 3D motifs joining canonical stems. It has become more and more apparent in recent years that the structural motifs in the loops, composed of noncanonical interactions, are essential for the final shape of the molecule enabling its multiple functions. Our capacity to predict accurate 3D structures is also limited when it comes to the organization of the large intricate network of interactions that form inside those loops. We previously developed the integer programming framework RNA Motifs over Integer Programming (RNAMoIP) to reconcile RNA secondary structure and local 3D motif information available in databases. We further develop our model to now simultaneously predict the canonical base pairs (with pseudoknots) from base pair probability matrices with or without alignment. We benchmarked our new method over the all nonredundant RNAs below 150 nucleotides. We show that the joined prediction of canonical base pairs structure and local conserved motifs (i) improves the ratio of well-predicted interactions in the secondary structure, (ii) predicts well canonical and Wobble pairs at the location where motifs are inserted, (iii) is greatly improved with evolutionary information, and (iv) noncanonical motifs at kink-turn locations. The source code of the framework is available at https://gitlab.info.uqam.ca/cbe/RNAMoIP and an interactive web server at https://rnamoip.cbe.uqam.ca/.

The Cryptic Nature of Fe-S Clusters: A Case Study of the Hepatitis B HBx Oncoprotein

Fe-S clusters are ubiquitous inorganic cofactors found in proteins across all domains of life, including viruses. Their prevalence stems from their unique redox and structural plasticity that supports functions ranging from electron transfer and catalysis to stabilization of protein structure. Although the ability of Fe-S clusters to exchange electrons is often functionally crucial, it can also act as an Achilles heel when these cofactors are exposed to oxidizing conditions, often leading to their degradation. This O2 sensitivity has rendered certain Fe-S clusters untraceable, particularly when the nascent proteins are isolated under ambient conditions. As a consequence of this O2 sensitivity, a growing number of proteins with roles in viral infection have been found to harbor Fe-S clusters rather than the annotated Zn2+ cofactor. The enigmatic protein X (HBx) of the Hepatitis B Virus is a multifunctional protein essential for viral replication and development of liver disease. Although HBx has defied biochemical characterization for over forty years, it has been shown to coordinate a redox-active Fe-S cluster that represents a significant feature for establishing its molecular function. The present review narrates the approaches to validate the HBx metallocofactor that can be broadly applied as a guide for uncovering the presence of Fe-S clusters in proteins with non-canonical sequence motifs.

Leishmania PNUTS discriminates between PP1 catalytic subunits through an RVxF–ΦΦ–F motif and polymorphisms in the PP1 C-tail and catalytic domain

PP1 phosphatases associate with specific regulatory subunits to achieve, among other functions, substrate selectivity. Among the eight PP1 isotypes in Leishmania, PP1-8e associates with the regulatory protein PNUTS along with the structural factors JBP3 and Wdr82 in the PJW/PP1 complex that modulates RNA polymerase II (Pol II) phosphorylation and transcription termination. Little is known regarding interactions involved in PJW/PP1 complex formation, including how PP1-8e is the selective isotype associated with PNUTS. Here, we show that PNUTS uses an established RVxF-ΦΦ-F motif to bind the PP1 catalytic domain with similar interfacial interactions as mammalian PP1-PNUTS and non-canonical motifs. These atypical interactions involve residues within the PP1-8e catalytic domain and N- and C-terminus for isoform specific regulator binding. This work advances our understanding of PP1 isoform selectivity and reveals key roles of PP1 residues in regulator binding. We also explore the role of PNUTS as a scaffold protein for the complex by identifying the C-terminal region involved in binding JBP3 and Wdr82, and impact of PNUTS on the stability of complex components and function in Pol II transcription in vivo. Taken together, these studies provide a potential mechanism where multiple motifs within PNUTS are used combinatorially to tune binding affinity to PP1, and the C-termini for independent binding of JBP3 and Wdr82, in the Leishmania PJW/PP1 complex. Overall, our data provide insights in the formation of the PJW/PP1 complex involved in regulating Pol II transcription in divergent protozoans where little is understood.

A Malassezia pseudoprotease dominates the secreted hydrolase landscape and is a potential allergen on skin

Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.

Endocytosis and Endocytic Motifs across the Connexin Gene Family.

Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

An ortho‐Quinone Methide Mediates Disulfide Migration in the Biosynthesis of Epidithiodiketopiperazines

AbstractThe transannular disulfide functions as a key structural element imparting diverse biological activities to epidithiodiketopiperazines (ETPs). Although mechanisms were proposed in previous studies, α,β′‐disulfide formation in ETPs is not well‐determined owing to the failure to identify the hypothetical intermediate. Herein, we characterize the key ortho‐quinone methide (o‐QM) intermediate and prove its involvement in the carbon‐sulfur migration from an α,α′‐ to an α,β′‐disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD‐dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of the α,β′‐disulfide was initiated by Gln140 triggering proton abstraction for generation of the essential o‐QM intermediate, accompanied by β′‐acetoxy elimination. Subsequent attack on the α,α′‐disulfide by Cys137 led to disulfide migration and spirofuran formation. This study expands the biocatalytic toolbox for transannular disulfide formation and sets the stage for the targeted discovery of bioactive ETPs.