Biology subjects

Christianson, D. W.

Publications and source records attributed to Christianson, D. W..

11 recordsLinked to original sources

bioRxiv2026

Binding of a Macrocyclic Nonapeptide Inhibitor to Histone Deacetylase 6

Histone deacetylase 6 (HDAC6) plays an essential role in the regulation of post-translational lysine acetylation. Myriad biological processes depend on the precise regulation of lysine acetylation-deacetylation cycles, and their dysregulation is linked to human pathologies such as cancer and neurodegeneration. Broad-specificity HDAC inhibitors are efficacious in treating disease, but the inhibitors currently approved for clinical use often cause deleterious side effects due to inhibition of multiple HDAC isozymes as well as other zinc enzymes. Cyclic peptides comprise novel platforms that, in principle, can be tailored for affinity and selectivity. A key advantage of cyclic peptide design is the ability to incorporate both canonical and noncanonical amino acids. As part of our program focusing on cyclic peptide HDAC inhibitors, we now report the 1.9-[A] resolution crystal structure of HDAC6 CD2 complexed with macrocyclic nonapeptide 1. The structure reveals zinc coordination by a noncanonical thiolate-containing amino acid, plus direct and water-mediated enzyme-inhibitor hydrogen bonds that contribute to nanomolar inhibitory potency. Structural comparisons with previously reported HDAC6 CD2 complexes with tetrapeptide and octapeptide macrocycles reveal similar networks of direct and water-mediated interactions. Given the surge of interest in cyclic peptide inhibitors, fueled in part by advances in computational design approaches, the crystal structure of the HDAC6 CD2-1 complex sets an important foundation for further development of macrocyclic peptide inhibitors.

biochemistry↗

bioRxiv2026

Improved Protein Semi-Synthesis Enables Biophysical Studies of Thioamide Destabilization of β-Sheet Interactions

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the {beta}-sheet region of the B1 domain of protein G (GB1) as well as {beta}-strand interactions in amyloid fibrils of the Parkinsons disease protein -synuclein. In both contexts, we observe dramatic destabilization of the {beta}-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of -synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

biochemistry↗

bioRxiv2025

Covalent Binding of Boronic Acid-Based Inhibitor GSK4394835A to Phosphodiesterase 3B, a Drug Target for Cardiovascular Disease

Boronic acids have become increasingly prominent in the pharmacopoeia ever since the US Food and Drug Administration approved Bortezomib (Velcade) for the treatment of multiple myeloma more than 20 years ago. Since then, four additional boronic acid-based drugs have been approved for clinical use in the treatment of cancer, fungal and bacterial infections, and eczema. The boron atom is inherently electrophilic due to being electron deficient, so the mode of inhibition by a boronic acid very often involves addition of a nucleophile, such as an enzyme-bound residue or a water molecule, to yield a tetrahedral boronate anion. Recently, a new class of boronic acid-based inhibitors of human phosphodiesterase 3B (PDE3B) have been reported, along with the 2.7 [A]-resolution crystal structure of PDE3B complexed with one of these inhibitors, GSK4394835A [Rowley et al. (2024) Discovery and SAR study of boronic acid-based selective PDE3B inhibitors from a novel DNA-encoded library. J. Med. Chem. 67, 2049-2065]. Surprisingly, the boronic acid moiety of the inhibitor was reported to bind as an intact, unreacted boronic acid. Additional discrepancies were evident in the reported refinement statistics, so we downloaded structure factor amplitudes from the Protein Data Bank (accession code 8SYC) and re-solved the structure. Our initial electron density maps clearly indicated nucleophilic addition of H737 to the boronic acid moiety of the inhibitor, generating a tetrahedral boronate anion in the active site. We re-refined the structure with excellent refinement statistics, concluding that GSK4394835A is a reversible covalent inhibitor of PDE3B.

biochemistry↗

bioRxiv2025

Structure and Mechanism of Avermitilol Synthase, a Sesquiterpene Cyclase that Generates a Highly Strained 6-6-3 Tricyclic Alcohol

Avermitilol synthase from Streptomyces avermitilis (SaAS) is a high-fidelity class I terpene cyclase that converts farnesyl diphosphate into a highly-strained, 6-6-3 tricyclic sesquiterpene alcohol. The mechanism of avermitilol formation proceeds through a 10-3 bicyclic intermediate, bicyclogermacrene, which undergoes proton-initiated anti-Markovnikov opening of two separate C=C bonds in a transannulation mechanism that forms the 6-6-3 tricyclic skeleton, with quenching by water to yield avermitilol. Small amounts of a side product, viridifloral, result from Markovnikov opening of one of the reactive C=C bonds. Here, we present enzymological studies of SaAS to establish the substrate scope and metal ion dependence for catalysis, and we present crystal structures of SaAS complexed with a variety of ligands that partially mimic carbocation intermediates in catalysis. Interestingly, these structures show that two water molecules remain trapped in a polar crevice in the active site regardless of the ligand bound. Structure-activity relationships for site-specific mutants yield key insight on the catalytic importance of these trapped water molecules. Specifically, T215 normally hydrogen bonds with water molecule W1, but the T215V substitution breaks this hydrogen bond and causes W1 to shift by 1.3 [A] to form a hydrogen bond with W300. Avermitilol generation is completely blocked in this mutant, but the generation of viridifloral and another side product is enhanced. Thus, the T215V substitution causes water molecule W1 to align for reaction with the tertiary and not the secondary carbon in the reactive C=C bond of bicyclogermacrene. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/675706v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@172e90corg.highwire.dtl.DTLVardef@1223258org.highwire.dtl.DTLVardef@1751d6org.highwire.dtl.DTLVardef@fdf739_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Cryo-EM Structure of the Cyclase Domain and Evaluation of Substrate Channeling in a Bifunctional Class II Terpene Synthase

Copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 [A]-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity.

biochemistry↗

bioRxiv2025

Crystal Structure and Catalytic Mechanism of Drimenol Synthase, a Bifunctional Terpene Cyclase-Phosphatase

Drimenol synthase from Aquimarina spongiae (AsDMS) is a highly unusual bifunctional sesquiterpene synthase that integrates two distinct, sequential isoprenoid processing activities within a single polypeptide chain. AsDMS catalyzes the class II cyclization of farnesyl diphosphate (FPP) to form drimenyl diphosphate, which then undergoes enzyme-catalyzed hydrolysis to yield drimenol, a bioactive sesquiterpene alcohol with antifungal and anticancer properties. Here, we report the X-ray crystal structures of AsDMS and its complex with a sesquiterpene thiol, which are the first of a terpene cyclase-phosphatase. The AsDMS structure exhibits a two-domain architecture consisting of a terpene cyclase {beta} domain and a haloacid dehalogenase (HAD)-like phosphatase domain, with two distinct active sites located on opposite sides of the protein. Mechanistic studies show that dephosphorylation of the drimenyl diphosphate intermediate proceeds through stepwise hydrolysis, such that two equivalents of inorganic phosphate rather than inorganic pyrophosphate are co-products of the reaction sequence. When the AsDMS reaction is performed in H218O, 18O is not incorporated into drimenol, indicating that the hydroxyl oxygen of drimenol originates from the prenyl oxygen of FPP rather than bulk water. These results correct a mechanistic proposal previously advanced by another group. Surprisingly, AsDMS exhibits unprecedented substrate promiscuity, catalyzing the conversion of substrate mimic farnesyl-S-thiolodiphosphate into cyclic and linear sesquiterpene products. Structural and mechanistic insights gained from AsDMS expand the functional diversity of terpene biosynthetic enzymes and provide a foundation for engineering "designer cyclases" capable of generating new terpenoid products.

biochemistry↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2025

Mechanism-Based Inhibition of Histone Deacetylase 6 by a Selenocyanate is Subject to Redox Modulation

Organoselenocyanates have attracted considerable attention in recent years due to their therapeutic potential and versatility in medicinal chemistry. Here, we report on the mechanism of inhibition by 5-phenylcarbamoylpentyl selenocyanide (SelSA-2), an analogue of the well-characterized histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, a.k.a. Vorinostat). We show that histone deacetylases 6 and 10 can promote selenocyanate hydrolysis to generate a selenolate anion, and we explore the redox chemistry of selenium as it modulates inhibitory activity through reversible formation of the diselenide. The 2.15 [A]-resolution crystal structure of histone deacetylase 6 cocrystallized with SelSA-2 conclusively demonstrates that it is not the selenocyanate, but instead the selenolate anion, that is the active pharmacophore responsible for enzyme inhibition.

biochemistry↗

bioRxiv2024

Structure of Bifunctional Variediene Synthase Yields Unique Insight on Biosynthetic Diterpene Assembly and Cyclization

An unusual family of bifunctional terpene synthases has been discovered in which both catalytic domains - a prenyltransferase and a cyclase - are connected by a long, flexible linker. These enzymes are unique to fungi and catalyze the first committed steps in the biosynthesis of complex terpenoid natural products: the prenyltransferase assembles 5-carbon precursors to form C20 geranylgeranyl diphosphate (GGPP), and the cyclase converts GGPP into a polycyclic hydrocarbon product. Weak domain-domain interactions as well as linker flexibility render these enzymes refractory to crystallization and challenge their visualization by cryo-EM. Despite these challenges, we now present the first experimentally-determined structure of a massive, 495-kD bifunctional terpene synthase revealing the assembly of all catalytic domains. The cryo-EM structure of variediene synthase from Emericella variecolor (EvVS) exhibits a bollard-like architecture, consisting of a hexameric prenyltransferase core sandwiched between two triads of cyclase domains. Although prenyltransferase and cyclase active sites are relatively close together, enzymological measurements indicate that GGPP is not channeled from one to the other. Surprisingly, however, the individual cyclase domain from another bifunctional diterpene synthase, fusicoccadiene synthase from Phomopsis amygdali, preferentially receives GGPP from the EvVS prenyltransferase in substrate competition experiments. Our previous studies of fusicoccadiene synthase suggest that GGPP channeling occurs through transient binding of cyclase domains to the sides of the prenyltransferase oligomer. The bollard-like architecture of EvVS leaves the sides of the prenyltransferase oligomer open and accessible, suggesting that a non-native cyclase could bind to the sides of the prenyltransferase oligomer to achieve GGPP channeling.

biochemistry↗

bioRxiv2024

Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis

Fusicoccadiene synthase from P. amygdala (PaFS) is a bifunctional assembly-line terpene synthase containing a prenyltransferase domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate (DMAPP) and three equivalents of isopentenyl diphosphate (IPP), and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are linked by a flexible 69-residue polypeptide segment. The prenyltransferase domain mediates oligomerization to form predominantly octamers, and cyclase domains are randomly splayed out around the prenyltransferase core. Previous studies suggest that substrate channeling is operative in catalysis, since most of the GGPP formed by the prenyltransferase remains on the protein for the cyclization reaction. Here, we demonstrate that the flexible linker is not required for substrate channeling, nor must the prenyltransferase and cyclase domains be covalently linked to sustain substrate channeling. Moreover, substrate competition experiments with other diterpene cyclases indicate that the PaFS prenyltransferase and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryo-EM structure of engineered "linkerless" construct PaFSLL, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the prenyltransferase octamer. Taken together, these results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the prenyltransferase octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

biochemistry↗

bioRxiv2023

Crystal Structure of Histone Deacetylase 6 Complexed with (R)-Lipoic Acid, an Essential Cofactor in Central Carbon Metabolism

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 [A]-resolution X-ray crystal structure of the HDAC6-(R)-dihydrolipoic acid complex, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-{pi} interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

biochemistry↗