Nonribosomal peptide synthetases produce diverse natural products using a multidomain architecture where the growing peptide, attached to an integrated carrier domain, is delivered to neighboring catalytic domains for bond formation and modification. Investigation of these systems can lead to the discovery of new structures, unusual biosynthetic transformations, and to the engineering of catalysts for generating new products. The antimicrobial β-lactone obafluorin is produced nonribosomally from dihydroxybenzoic acid and a β-hydroxy amino acid that cyclizes into the β-lactone during product release. Here we report the structure of the nonribosomal peptide synthetase ObiF1, highlighting the structure of the β-lactone-producing thioesterase domain and an interaction between the C-terminal MbtH-like domain with an upstream adenylation domain. Biochemical assays examine catalytic promiscuity, provide mechanistic insight, and demonstrate utility for generating obafluorin analogs. These results advance our understanding of the structural cycle of nonribosomal peptide synthetases and provide insights into the production of β-lactone natural products. The antimicrobial β-lactone obafluorin is produced by a Nonribosomal Peptide Synthetase (NRPS). Here the authors present the crystal structure of the obafluorin NRPS and develop a reconstitution assay that allows them to analyse product formation from obafluorin NRPS mutants and alternate substrates. It has been suggested that ~70% of potential biosynthetic gene clusters (BGCs) differ significantly from well characterized BGCs7, providing an impetus for identifying the structural features of enzymes that catalyze the formation of unusual natural products, including those harboring a β-lactone group.β-Lactones are endowed with a highly strained ring system that affords these compounds a unique reactivity among natural products. In particular, β-lactones are well-suited for the inhibition of hydrolases, transferases, ligases, and oxidoreductases8. During the biosynthetic cycle, the nascent peptide is bound as a thioester to a peptidyl-carrier protein domain  domain) through a phosphopantetheine (pPant) cofactor and delivered to different catalytic domains for extension and modification10. The core catalytic domains of NRPSs are the adenylation (A domain) that loads the selected amino acid, the condensation (C domain) that carries out peptide bond formation, and the thioesterase domain (TE domain) that catalyzes release of the mature peptide through hydrolysis or macrocyclization8.One class of natural products are peptides produced by the nonribosomal peptide synthetase (NRPS) enzymes. NRPSs contain multiple catalytic domains joined in one or more multidomain proteins that operate in an assembly line fashion to produce a peptide from standard and nonproteinogenic amino acids11, EntF12, and the uncharacterized NRPS AB340312, as well as the initiation module of LgrA13 and the cross-module structure of the DhbF14, offer clues into the conformational dynamics that drive a three-state structural cycle12. In state 1, illustrated by the AB3403 protein, the adenylation domain adopts a conformation that catalyzes the initial adenylation of the amino acid substrate12. In state 2, a small C-terminal subdomain of the A domain rotates by 140° to accompany the delivery of the downstream PCP domain to the A domain pocket for loading of the pPant cofactor15. This thioester-forming conformation has been observed in the structure of EntF12, as well as DhbF structure14. Upon loading of the substrate on the PCP domain, the adenylation C-terminal subdomain reverts to state 1, a conformation in which the loaded substrate is delivered to the C domain for peptide bond formation with an upstream substrate. Simultaneously, the A domain adopts the adenylate-forming conformation to enable activation of another substrate molecule for a subsequent round of extension. Finally, the extended peptide substrate on the PCP is delivered to downstream C or TE in state 3, which has not been observed structurally in the context of a complete module but which can be inferred from existing didomain structures of PCP-TE16 or of PCP-C17.Structural studies of NRPS modules offer insight into the features that govern the efficient delivery of the substrate to the various catalytic domains. The structures of termination modules SrfA-C19 has identified the key steps in the biosynthesis of obafluorin, a nonribosomal peptide from Pseudomonas fluorescens (ObiF/D) and Burkholderia diffusa  to the α-amino group of -β-hydroxy-p-nitro-homophenylalanine (β-OH-p-NO2-homoPhe) via amide bond linkage with accompanying 4-membered lactone formation via the β-hydroxy group. The unusual β-hydroxy-α-amino acid is derived from a crossed aldol reaction of a glycine enolate generated from l-Thr with p-nitro-phenylacetaldehyde (PNPAA) catalyzed by the PLP-dependent l-Thr transaldolase, ObiH. The NRPS proteins are distributed across two (ObiF/D in P. fluorescens) or three (ObiF1/F2/D in B. diffusa) polypeptide chains . The C-terminal adenylation domain (AAr) activates 2,3-DHB as the corresponding acyl adenylate and loads a free-standing aryl carrier protein (PCPAr) ObiD. The A-domain embedded in ObiF activates β-OH-p-NO2-homoPhe and loads the adjacent PCP domain. The C domain is presumably responsible for amide bond formation while the TE domain catalyzes cyclization to the β-lactone and product release, enabling turnover of the ObiF catalytic cycle. Interestingly, in B. diffusa, the AAr domain for 2,3-DHB is a free-standing protein (ObiF2) that interacts in trans with the multidomain C-A-PCP-TE-MLP NRPS module (ObiF1).Recent work24. In ObiF from P. fluorescens, the MLP is positioned between the TE and terminal AAr domain. In contrast, the NRPS from B. diffusa (ObiF1) terminates at the MLP.The obafluorin NRPS contains several other surprising features. First, the TE domain, containing a less common catalytic cysteine, is responsible for both β-lactone formation and peptide release. In addition, ObiF contains an integrated MbtH-like protein (MLP), a small domain that has been shown to interact with some adenylation domains to improve protein solubility, or to alter A domain activity or possibly specificity18, we present the X-ray crystal structure of holo-ObiF1 from B. diffusa. The structure shows that the MLP domain interacts with the upstream, non-adjacent adenylation domain, an interaction that to our knowledge has not previously been observed. We identify the catalytic triad in the ObiF1 TE domain that harbors the cysteine residue and also an aspartic acid residue on β-strand 6, a less common position for NRPS thioesterase domains. An in vitro reconstitution assay validates the importance of this MLP interaction, identifies an arginine residue that may be involved in inter-domain acyl transfer from the PCP thioester to the TE domain catalytic nucleophile, and shows that the unusual triad configuration is exploited to enable β-lactone cyclization within the TE domain. Finally, we assessed the substrate scope for the B. diffusa enzymes to provide insight into A-domain selectivity and capacity for analog production. The functional and structural studies reported here are consistent with direct β-lactone ring closure catalyzed by the TE domain. Together these results extend our understanding of the structural cycle of the NRPS enzymes and highlight several unanticipated features that should facilitate the identification of more β-lactone-containing natural products.To identify the structural features that govern NRPS-catalyzed obafluorin biosynthesis and to further establish the mechanism outlined previouslyholo-ObiF1  inhibitor to covalently tether the PCP domain pPant arm to the A domain, the ObiF1 structure features a conformation in which the pPant arm of the thiolation domain is docked into the condensation domain cleft. We compared the structure of ObiF1 with previously characterized NRPS enzymes, including large multidomain structures as well as single domain enzymes  of iF1 Fig. . SimilarThe most striking difference of the ObiF1 structure and previous examples of NRPS modules is that the N-terminal portion of the terminal MLP domain is threaded through the interface formed between the A and C domains to associate with and activate the A domain Fig. . This doN-acyl-α-amino-β-lactones, we compared the structure of the ObiF1 embedded A domain with the crystal structure of the l-Thr activating A domain Thr1 (5N9X), which is involved in 4-chlorothreonine biosynthesis in Streptomyces sp. OH-509327. The 2S, 3R stereocenters within the β-OH-p-NO2-homoPhe backbone are set simultaneously during the ObiH-catalyzed trans-aldol reaction 30 with a few key differences that accommodate the expanded steric bulk of the p-NO2-homoPhe side chain relative to that of l-Phe. The specificity-conferring residues of ObiF1 form the hydrophobic pocket for the p-NO2-phenyl group of the substrate and make no interactions with the polar or charged functional groups along the substrate backbone. Cys734 is located in the pocket and potentially could interact with the NO2 moiety; however, as currently modeled the thiol is directed away from the substrate. The α-amino group hydrogen bonds to the amide proton of Thr766 and forms a salt-bridging interaction with Asp662. Finally, Ile765 is conserved between both ObiF1 and Thr1 and may play a role in promoting β-strand dihedral angles that orient the Gly764 carbonyl toward the β-OH-p-NO2-homoPhe β-hydroxyl group, thus promoting H-bond formation.The specificity-conferring sequence of the ObiF1 A domain is D/A/W/G/C/G/M/I Fig. , which i23. While many MLPs are expressed as free-standing proteins, some proteins, including SlgN131 and NikP132, possess an MLP that is appended to the N-terminus of its partner A domain. With SlgN1 or NikP1 serving as a model, the presence of the MLP domain in the P. fluorescens ObiF protein between the TE domain and the C-terminal AAr domain suggested a similar interaction with the immediate downstream domain. With the separate ObiF1 and ObiF2 of B. diffusa, this domain architecture raised the possibility that the MLP might interact in trans with the free-standing ObiF2 AAr domain. Surprisingly, the C-terminal MLP binds to the upstream A domain of ObiF1  to disrupt this interaction. We also generated a truncated construct (ObiF1-del1304) in which the protein was terminated after the TE domain. We expressed this protein alone, or with a tandem expression system to co-express the ObiF1-del1304 protein with the ObiF1  MLP domain, residues 1304 - 1390 in trans . Upon purification the yield of these three protein constructs, A841E, ObiF1-delMLP, and rbs1303/4, was much lower than WT ObiF1. Analysis of the final size exclusion chromatography purification steps demonstrated that the three proteins with missing or disrupted MLP interaction were much more likely to aggregate and elute in the column void volume -β-OH-homoPhe PCP thioester. There are three mechanistic possibilities for product cleavage from the final PCP thioester intermediate, including solvolysis by glutathione (GSH) in the reaction buffer (path a), acyl transfer to the TE domain followed by beta-lactone formation (path b), or hydrolysis (path c)  compared with the parent β-lactone (t1/2 ~ 5 min at pH 7), making thioesters a useful proxy for quantifying β-lactone formation18.We developed a simple in vitro reconstitution assay for ObiF1/F2/D/H using LCMS to quantify product ion counts Fig. . The adv c) Fig. . Any β-lE. coli deficient in native MLP production, this implies that the NRPS is still functional, albeit at reduced efficiency, without an MLP interaction. Increasing MLP concentrations to 10 or 25 μM did not increase product ion counts suggesting saturation of the MLP-binding site at stoichiometric levels of added MLP relative to ObiF1-del1304. Coexpression of the ObiF1 MLP domain with the del1304 mutant from the same pET28 plasmid produced protein sample rbs1303/4 that gave enhanced product ion counts relative the del1304 mutant alone, but still at approximately fourfold reduced levels to WT. Similar to the del1304 mutant, addition of exogenous MLP recovered product ion counts for the rbs1303/4 protein reaction to WT levels. Collectively, the results from MLP supplementation of del1304 and rbs1303/4 support the notion that in trans MLP interactions compensate for deletion of the C-terminal MLP domain in ObiF1.Product formation by 1 μM WT ObiF1 was not affected by the presence of exogenous MLP domain at 1, 10, or 25 μM Fig. . This isAr ObiF2. The MLP-related mutant activities in the functional assay support interaction of the MLP domain with the ObiF1 module as observed in the crystal structure. However, it does appear that tethering the MLP domain to the NRPS module is not required suggesting the MLP domains acting in trans for related NRPSs may have interactions with upstream A domains embedded in NRPS modules.Similar to the del1304 mutant, the A841E mutant generated ~25-fold lower product ion counts compared with WT ObiF1 . Contrary to del1304, however, addition of 1 μM MLP provided only a modest enhancement in product ion counts. Increasing the concentration of added MLP to 10 and 25 μM further enhanced product ion counts without ever reaching WT product levels (still approximately eightfold lower than WT at 25 μM MLP). The apparent dose dependence of MLP-promoted product formation for the A841E mutant is consistent with a reduced binding affinity of the MLP domain at the modified MLP-binding site. To gain insight on the effect of MLP on ObiF2 activity, we checked levels of the 2,3-DHB acyl adenylate by LCMS for reactions with WT, del1304, rbs1303/4, and A841E ObiF1 variants  catalytic cysteine is required for β-lactone formation18. We observed a hydrogen bonding network of the catalytic triad at residues Cys1146, His1284, and Asp1254 , a type II TE domain with acyl-PCP hydrolase activity , possibly to minimize steric clash with the substrate-loaded pPant arm during acyl transfer to Cys1146 of the TE domain active site.The ObiF TE domain -β-OH-homoPhe PCP thioester blocking transfer to the TE domain and leaves only solvolysis pathways a and c outlined in Fig. After the PCP domain is successfully loaded with the intermediate To examine the apparent preference of ObiF1 for a RifR-like over an EntF-like triad configuration we generated a ObiF1 double mutant with an EntF-like positioning of the aspartic acid residue (G1173D/D1254A). This double mutant displayed a reduced activity relative to the D1254A single mutant in the ObiF1/F2/D/H reconstitution assay, as evidenced by an approximately fivefold reduction in product ion counts Fig. . The attTo explore other residues that might be involved in substrate transfer from the PCP to TE domains, we prepared Ala mutants of Asp1177, Arg1263, and Arg1190 residues around the ObiF1 TE active site. The D1177A and R1263A mutants generated product ion counts equivalent to WT ObiF1, while the R1190A mutation resulted in a ~10-fold reduction in product ions. Product levels for the R1190A mutant were still ~16-fold higher than the C1146A mutant suggesting that substrate transfer to the TE domain is still possible at reduced efficiency relative to WT ObiF1. We note that we employed phenylacetaldehyde (PAA) in the ObiF1/F2/D/H reconstitution assay in both WT control and mutant reactions; therefore, any potential ionic interactions between the Arg1190 side chain and the obafluorin nitro group can be excluded as a contributing factor in substrate pre-organization.1a–h) and benzoic acid derivatives (3a–u) in the ObiF1/F2/D/H reconstitution assay . For reactions with variable benzoic acid substrates, the aldehyde substrate was always PAA (1a). Product nomenclature reflects the identity of the aldehyde (first letter) and benzoic acid (second letter) substrates. We again used the thiol capture approach for quantifying β-lactones, this time using dithiothreitol (DTT) as the reactive thiol nucleophile. The use of DTT resulted in initial formation of the DTT-thioester that rearranges to the more thermodynamically stable DTT-oxoester that does not undergo further hydrolysis under the reaction conditions , thioesters (5), and N-acyl-β-hydroxy acids (6) that were normalized to a phenylalanine internal standard. All product identities were confirmed using high-resolution mass spectrometry  are converted to the corresponding β-hydroxy-α-amino acids (2a–h) by transaldolase ObiH to provide a steady equilibrium concentration of the NRPS substrate that can only be driven toward N-acylated products by ATP consumption. In most cases, the β-hydroxy-α-amino acids (2a–h) can be detected by LCMS  are not detectable, we still observed product formation . There are also instances where the amino acid substrate was detected at early time points and was fully consumed over the course of 24 h, presumably due to complete conversion of the starting aldehyde (the limiting reagent) to thioester (5) and β-hydroxy acid (6) products . The best substrates for ObiF1 were β-hydroxy-α-amino acids  derived from phenylacetaldehydes that most closely resemble the natural PNPAA aldehyde substrate that gives rise to p-nitro-substituted obafluorin  at low levels, but no NRPS products were detected indicating that the ObiF1 A domain poorly activates these substrates. Products resulting from linear alkyl  and extended aryl  substrates were also generated. This indicates that the phenyl ring or a hydrophobic alkyl chain must extend deep into the ObiF1 A domain substrate binding pocket in order to avoid unfavorable steric clashes, as indicated by the crystal structure showing active site occupation by the natural substrate β-OH-p-NO2-homoPhe  the corresponding (E)-phenethylenyl side chain was not activated by ObiF1 indicating that substrate flexibility plays a role in A domain binding and/or activation.The aldehydes , gave the highest product ion counts  and a clear preference for 2,3-substitution of the benzoic acid derivative emerged when comparing product ion counts for the 21 substrates that were included in the study. For example, 2-OH and 3-OH benzoic acids (3c and 3d) were both activated and converted efficiently to products 5ac/6ac and 5ad/6ad, respectively; while 4-OH benzoic acid gave significantly lower product ion counts only detectable for the thioester 5ae. Some halogen substituents (Cl and F) at the 2,3-positions were accepted as substrates, but this appeared to be somewhat dependent on atomic radius. Larger halogens, Cl and Br, at the 4-positions gave no product ion counts and 2,3-Cl (an) products were not detected while 2,3-F (aq) product ions were strong. We checked benzoic acid acyl adenylate levels by LCMS and found 100% correlation between the presence of acyl adenylate and the presence of product ions in the enzymatic reactions  . However, there were some instances where acyl adenylate was detected, but no product ions were formed  and the amount of acyl adenylate ions did not always correlate with the amount of product ions, which is to be expected since each analog will have variable stability and ionization potential under the assay conditions. This implies that product formation and scope of analog production is determined by the adenylation selectivity of ObiF2 for benzoic acids and the domain specificity in the NRPS module ObiF1 for structurally modified intermediates.The Ates Fig. . The natN-acyl-α-amino-β-lactone formation from the ObiF1 NRPS system. ObiF1 has a unique MLP domain that forms a non-local protein-protein interaction with the upstream A domain. The ObiF1 A domain tolerates a variety of bulky hydrophobic β-hydroxy-α-amino acids while excluding activation of the proteinogenic β-hydroxy-α-amino acids l-Ser and l-Thr. ObiF1 is somewhat unusual in that its resulting β-lactone is the cis stereoisomer, a consequence of the β-OH-p-NO2-homoPhe backbone stereochemistry which is set during the ObiH-catalyzed cross-aldol reaction18. The ObiF1 A domain is likely constrained to activate β-hydroxy-α-amino with the same stereochemistry as β-OH-p-NO2-homoPhe. β-Hydroxy-α-amino acids with configurations similar to that of allo-l-Thr would likely be unable to participate in the favorable backbone carbonyl H-bond network observed in the A domain active site and thus disfavored as an ObiF1 substrate.Here we describe the structural basis that defines the genetic signatures of 37, this observation illustrates that internal MLPs can also interact beyond an immediately downstream A domain. Like other MLPs, the domain from ObiF1 enhances protein stability and function, even in light of the new structural constraints that it adds to the modular conformation.We observed an unexpected interaction of the C-terminal MLP domain with the upstream A domain in the ObiF1 protein. This interaction was validated biochemically and appears to be a new type of interaction. While it has previously been demonstrated that free-standing MLPs can interact with multiple NRPS A domains within a biosynthetic pathwayexo-trig ring closure to generate the β-lactone. Acyl transfer to the TE domain ensures that the thioester is protected from solvolysis and enters the TE domain active site environment that promotes kinetically favorable β-lactone ring formation, probably with conformational control leading to stabilization of the 4-exo-trig cyclization transition state. This dominant hydrolysis of the C1146S mutation results from the enhanced reactivity of a thioester versus an oxoester with a hydroxyl nucleophile, which was also noted in the increased promiscuity of an engineered TE domain from the pikromycin polyketide synthase system38. The cysteine thiolate is generated by deprotonation of the Cys1146 thiol via His1284 and Asp1254, and removal of either side chain significantly diminishes ObiF1 activity. Surprisingly, the ObiF1 TE domain positions the catalytic Asp on β-strand 6 in a configuration that is similar to the type II TE domains RifR and RedJ. The EntF-like triad configuration (Asp on β-strand 5) sterically inhibits substrate loading onto Cys1146. Acyl transfer from the pPant arm to the TE domain cysteine competes with solvolysis by environmental thiols and water; perhaps the larger lid-loop region of the ObiF TE domain and the RifR-like triad configuration allow less solvent into the active site compared with TE domains in which the only function is substrate release. Both EntF and ObiF1 NRPSs generate N-2,3-DHB-β-hydroxy-α-amino acid intermediates that differ only in the identity of the β-hydroxy-α-amino acid substrate, l-Ser and β-OH-p-NO2-homoPhe, respectively. The similarity in NRPS domain orientation between ObiF1 (C-A-PCP-TE-MLP) and EntF (C-A-PCP-TE) highlights the importance of the alternate catalytic properties of NRPS TE domains in determining product outcome from the thioester tethered N-2,3-DHB-β-hydroxy-α-amino acid intermediates, iterative trimerization and macrolactonization for EntF and β-lactone formation for ObiF1.The GXCXG TE domain motif appears to facilitate β-lactone formation via two related processes which include acyl transfer  to the active site cysteine thiolate followed by 4-The ObiF1 TE domain possesses some general features that might facilitate β-lactone formation. These features include a large lid loop that occludes solvent from the active site Cys1146 residue and a catalytic triad configuration similar to that observed in the type II TE domains RifR and RedJ. Presumably, this configuration allows adequate space for a hydrophobic pocket to form adjacent to the Cys1146 side chain. This pocket reduces the conformational degrees of freedom available to the tethered substrate, thus promoting cyclization and subsequent product release. Expansion of this active site pocket via mutation may further expand the substrate scope of the ObiF TE domain.B. diffusa provides an improved understanding of NRPS domain interactions, substrate selectivity, and termination chemistry. We have established the structural blueprint for a β-lactone forming NRPS facilitated by a unique TE catalytic domain that kinetically favors formation of the strained 4-membered ring product. Our findings can be applied toward genome mining efforts, chemoenzymatic syntheses, and mechanistic studies of related NRPS and PKS biosynthetic assembly lines that generate strained β-lactone and β-lactam 4-membered heterocycles.Our structural and functional characterization of the ObiF1/F2 NRPS assembly line from Burkholderia diffusa gene encoding for ObiF1  was synthesized, codon optimized . In total, 1.5 L of TB media (30 μg/mL kanamycin) was inoculated with 1.5 mL of an LB (30 μg/mL kanamycin) overnight culture  of E. coli BL21(DE3) cells harboring the pET28-ObiF1 plasmid. The 1.5 L TB culture was grown to an OD600 of ~0.7  at which time IPTG (500 μM) was added and the culture was incubated for 18 h . Cells were harvested via centrifugation  and flash frozen in liquid nitrogen. Subsequent purification steps were performed at 4 °C. Cells were re-suspended in lysis buffer .The 2 (1 mM), and five equivalents of coenzyme A (400 μM). The pantetheinylation reaction was then rocked gently for 3 h at 4 °C after which the reaction was further purified by gel filtration  in 50 mM HEPES pH 8.0, 500 mM NaCl, 0.2 mM TCEP, 10% v/v glycerol. Finally, ObiF1 protein was exchanged into a crystallization buffer composed of 25 mM HEPES pH 8.0, 25 mM NaCl, 0.4 mM TCEP, 5% v/v glycerol . Concentrated ObiF1 protein solution (36.5 mg/mL) was flash frozen in liquid nitrogen and stored at −80 °C , and the resulting cell lysate was clarified via ultracentrifugation , filtered (0.45 μm), and passed over a Ni-loaded HisTrap column (2 × 5 mL columns in series). The column was washed with 2.5 column volumes of 10% v/v elution buffer  in lysis buffer and immobilized protein was eluted with 3 column volumes of elution buffer. Protein-laden fractions were pooled (11.9 mg/mL protein) and combined with the promiscuous phosphopantetheinyl transferase Sfp (400 nM), MgClInitial crystallization conditions were identified through the Hauptman-Woodward Medical Research Institute High-throughput Screening (HTS) Laboratory. Preliminary crystals, used to obtain an initial set of phases, were grown using the microbatch-under-oil technique; crystallization droplets were setup under paraffin oil in an untreated, polystyrene 72 well microbatch plate from Hampton Research  at 23 °C. Crystallization droplets (2 μL) were composed of a 1:1 ratio of ObiF1 protein solution and a crystallization cocktail .These preliminary crystals were cryo-protected by treatment with mixtures of the crystallization cocktail, an aqueous sucrose solution (50% w/v), and cryo-solution A using the following procedure. Cryo-solution A was made by combining ethylene glycol (neat) and an aqueous solution of non-detergent sulfobetaine 201  in a 1:9 ratio. Cryo-solution B was made by combining cryo-solution A:crystallization cocktail:sucrose solution in a 10:3:7 ratio. Preliminary crystals were soaked for ~15 s in two droplets (~5 μL) composed of 25% and 50% v/v cryo-solution B diluted in crystallization cocktail, in order of increasing cryo-solution B concentration, followed by vitrification in liquid nitrogen.holo-ObiF1 protein solution at 36 mg/ml and 1 µL of crystallization cocktail supplemented with 2 mM of β-OH-p-NO2-L-homoPhe. Purified β-OH-p-NO2-L-homoPhe (> 98% pure) was prepared enzymatically as described previously18. Briefly, p-NO2-phenylpyruvate (5 mM) was treated with decarboxylase ObiG (10 μM) and transaldolase ObiH (10 μM), both from P. fluorescens ATCC 39502, in a buffer containing ThDP (10 μM), MgCl2 (10 μM), PLP (10 μM), l-Thr (5 mM), and MES (25 mM) adjusted to a final pH of 7.5. All concentrations reflect the final concentration in a 1 mL total reaction volume. After 3 h at room temperature, the reaction was quenched by addition of TFA and pure was β-OH-p-NO2-L-homoPhe was obtained after purification by preparatory HPLC  100A column; mobile phase (A) was 0.1% TFA in water; mobile phase (B) was 0.1% TFA in MeCN; gradient was 0% B held for 5 min then 0% B to 100% B over 10 min; retention time ~14 min). Crystals were cryo-protected by serial transfer through four drops  of 70 mM HEPES pH 7.0, 8 mM NaCl, 1.7% v/v glycerol, and 1.7% w/v 1,3-dimethylimidazolium dimethyl phosphate with increasing PEG3350 concentration . Each drop was supplemented with 2 mM β-OH-p-NO2-homoPhe. Following the final soak in the 45% w/v PEG3350 solution, crystals were vitrified directly in liquid nitrogen.The microbatch-under-oil crystallization procedure was further optimized. Crystals were grown at 14 °C and the crystallization droplet comprised 2 µL of P212121 with the XDS program package. The ligand-bound dataset was indexed and integrated in the DIALS program package followed by scaling and merging in space group P212121 with AIMLESS. Due to the moderate diffraction anisotropy, processed intensities were converted to structure factor amplitudes with the program phenix.reflection_file_converter using the “massage_intensities” feature, which employs a flat prior probability distribution during the conversion of negative intensities to positive amplitudes. The ObiF1 ligand-bound model was refined against these processed amplitudes.Diffraction data were collected at APS beamline 23-IDD  and SSRL beamline 12-2 . Each diffraction dataset (3.0 Å) was derived from a single crystal and exhibited moderate anisotropy. Diffraction data for the unliganded dataset were indexed, integrated, scaled, and merged in space group 5U89), the condensation domain showed 28% identity to condensation domain of EntF (5T3D), and the thioesterase domain showed 29% identity to the type II thioesterase from the rifamycin biosynthetic cluster (3FLB). The domains were placed in order from the largest to the smallest domains. A search model for each domain was generated using the PHENIX Sculptor module to remove non-conserved side chains and gaps.An initial structure was determined from the unliganded dataset through iterative molecular replacement with PHASER, as implemented in the PHENIX suite of programs. A search of the Protein Data Bank identified structures with high sequence homology to each domain of ObiF1. Specifically, the adenylation domain showed 39% sequence identity to the adenylation domain of DhbF (The adenylation domain was first placed via molecular replacement using PHASER. This was used as a fixed model in the subsequent search for the condensation domain. The two domains were positioned similar to the didomain core of prior NRPS modules. Placement of the thioesterase domain provided a convincing model and map. No effort was made to place the PCP or MbtH-like protein (MLP) domain through molecular replacement. The final molecular replacement solution of all the three domains contained 1165 residues. Removal of residues with poor density identified a core conserved 769 residues that served as a starting point for manual model building and refinement. Continued building of loops and side chains led to improved electron density, which enabled the identification of the remaining domains.sub domain (924:928). Disorder within these regions is consistent with the dynamic domain configuration that is required to enable the PCP domain to visit the neighboring catalytic domains within the module.The domains of ObiF1 , C domain (residues 5–444), and TE domain (residues: 1059–1303). The PCP domain and resolvable linker regions were then built in manually with iterative real space refinement in Coot. Restraints for β-OH-p-NO2-L-homoPhe and phosphopantetheine were generated with eLBOW39. The liganded ObiF1 structure was further refined with PHENIX refine. Final rounds of refinement employed translation-libration-screw (TLS) parameterization; 9 TLS domains defined at residues 4:169, 170:379, 380:453, 454:616, 617:776, 777:870, 1059:1131, 1132:1303, and 1313:1370 were determined with the TLSMD server40. Initially, 12 TLS domains were determined with the TLSMD server; three of these initial domains corresponding to the A domain C-terminal subdomain and thiolation domain were excluded from TLS refinement. Final refined TLS matrices were confirmed to correspond to harmonic motion of rigid bodies with the program PHENIX tls_analysis41. Diffraction and refinement statistics are displayed in Supplementary Table Protein atoms from the unliganded ObiF1 protein were used as a molecular replacement search model to determine the structure of the protein bound to the β-OH-obiF1 mutants encoding A841E, C1146A, C1146S, G1173L, D1177A, R1190A, D1254A, R1263A, H1284A, D1254A/H1284A, and G1173D/D1254A proteins. In addition to the A841E mutant, we designed two additional constructs to validate the interaction of the ObiF1 MLP and adenylation domains. A truncated del1304 protein that lacked the entire MLP domain was produce by mutagenesis to introduce a stop codon after the codon for Leu1303. A second construct designated rbs-1303/1304 introduced a stop codon, a ribosome binding site, and an ATG codon between the TE and MLP domains to co-express the ObiF1 C-A-PCP-TE and MLP proteins. Finally, we designed the ObiF1-MLP-1315 construct to produce the ObiF1 MLP protein consisting of residues Ala1315–Val1390 for supplementation assays. Following the Val1390 residue, this construct encoded a purification tag with the amino acid sequence of KLAAALEHHHHHH.PCR-based mutagenesis was used to produce 42 was employed for mutants rbs-1303/4 and del1304. For these two mutants, separate PCR reaction mixtures (50 µL) were setup for both forward and reverse primers (400 nM each). The reactions were run for one cycle, combined , and then run for an additional 29 cycles.Mutagenesis primers were produced by Integrated DNA Technologies . Clones were selected for on LB agar containing kanamycin (50 μg/mL). For double mutants D1254A/H1284A and G1173D/D1254A, mutagenesis PCR and transformation procedures were performed iteratively using pET28-ObiF1-D1254A as a template for the second reaction. ObiF1-MLP-1315 was PCR amplified from a pET28-ObiF1 template and cloned into a pET24 vector (NdeI/HindIII) with a C-terminal 6xHis-tag. Plasmids were then isolated and sequenced to establish the presence of the desired mutation(s). Sequence-confirmed plasmids were then used to transform competent BL21(DE3) E. coli cells for protein expression. The ObiF1-WT and TE domain mutants  were produced in BL21(DE3) for purification. In contrast, the wild-type ObiF1, the ObiF1 MLP protein, and mutant ObiF1 enzymes used to test the role of the MLP interaction  were expressed in BL21(DE3)ΔybdZ , which lacks the only endogenous E. coli MLP gene.Following completion of PCR amplification, DpnI (0.5 μL) was added to reaction mixtures, which were incubated at 37 °C for ~1 h. PCR products were purified by gel electrophoresis (0.9–1% w/v agarose) and then transformed into NEB Turbo competent E. coli BL21(DE3) cells harboring WT or mutant plasmid. The TB culture was grown  until the culture reached an OD600 of ~0.6 at which time IPTG (500 μM) was added and the culture was incubated for 20 h . Cells were harvested via centrifugation  and flash frozen in liquid nitrogen. Subsequent purification steps were performed at 4 °C. Frozen cell pellets were thawed and re-suspended in lysis buffer . Cells were lysed by mechanical disruption (Branson Sonifier) and the resulting cell lysate was clarified via centrifugation , filtered (0.45 μm), and passaged over a Ni-loaded HisTrap (5 mL) column. The column was washed with 5 column volumes of 10% v/v elution buffer  in lysis buffer and immobilized protein was eluted with 6 column volumes of elution buffer. Fractions containing ObiF1 were combined and pantetheinylated as described (vide supra). Pantetheinylation reactions were then purified via gel filtration . Gel filtration of ObiF1-WT was carried out in 50 mM KH2PO4 pH 8.0, 150 mM NaCl, and 0.2 mM TCEP. Gel filtration of ObiF1 TE domain mutants was carried out in 50 mM KH2PO4 pH 8.0, 500 mM NaCl, 0.2 mM TCEP, and 10% v/v glycerol. Following gel filtration, fractions containing ObiF1 mutants were pooled  and dialyzed (12–14 kDa MWCO) for 20 h against 1 L of 50 mM KH2PO4 pH 8.0, 150 mM NaCl, and 0.2 mM TCEP. Following gel filtration (ObiF1-WT) or dialysis (ObiF1 TE domain mutants), samples were concentrated to 21–28 mg/mL , flash frozen in liquid nitrogen, and stored at –80 °C.The wild-type ObiF1 and TE domain mutants  were purified using a slightly different protocol than the ObiF1 used for structural studies. One liter of TB media (30 μg/mL kanamycin) was inoculated with 1 mL of an LB (30 μg/mL kanamycin) overnight culture  of E. coli BL21(DE3)ΔybdZ cells harboring WT or mutant plasmid. Subsequent expression and purification steps were performed as described for ObiF1 TE domain mutants (cell pellets harboring ObiF1-MLP-1315 were re-suspended in 10 mL lysis buffer per gram cell pellet). Following gel filtration, fractions containing the desired protein were pooled, concentrated directly in gel filtration buffer , flash frozen in liquid nitrogen, and stored at –80 °C.Wild-type ObiF1 and the ObiF1 MLP domain mutants , as well as the free-standing MLP (ObiF1-MLP-1315) were purified for in vitro enzyme reactions. One liter of TB media  was inoculated with 1 mL of an LB  overnight culture  of N-terminal hexahistidine tag . ObiF2, ObiD, and ObiH were expressed with N-terminal hexahistidine tags from 1 L cultures of E. coli BL21 (DE3) in TB medium supplemented with 50 μg/mL kanamycin. Expression cultures were inoculated from a 5 mL overnight culture of E. coli BL21 (DE3) harboring the appropriate plasmid and grown to OD600 ~0.4. Cultures were then cooled in an ice bath prior to addition of a sterile IPTG solution . Cultures were then incubated at 15 °C with agitation for ~18 h. Cells were cooled to 4 °C  and harvested via centrifugation at 5000 rpm for 20 min. The supernatant was discarded and cell pellets were suspended in 40 mL of lysis buffer  and flash frozen in liquid nitrogen. After thawing, cells were lysed by two passages through an Avestin EmulsiFlex-C5 cell disruptor. Cell lysate was clarified via centrifugation at 45,000 rpm for 35 min. Supernatant was treated with Ni-NTA resin for 30 min. After two washings with lysis buffer the proteins were eluted with 5 × 10 mL aliquots of elution buffer . Elution fractions were analyzed by SDS-PAGE with Coomassie blue visualization. Fractions containing pure proteins were combined and dialyzed into phosphate buffer  using 10,000 MWCO SnakeSkin dialysis tubing. Protein solutions were concentrated using Amicon 10,000 MWCO centrifugal filters followed by flash freezing as ~50 μL beads and storage at –80 °C.Genes encoding ObiF2 (WP_059467197.1), ObiH (WP_059467200.1), and ObiD (WP_059467195.1), were synthesized, codon optimized , and the benzoic acid substrate was 2,3-DHB (3a). For all ObiF1 substrate screens the thiol trapping agent was DTT. For aldehyde (1a–h) substrate screens the benzoic acid substrate was 2,3-DHB (3a). For benzoic acid substrate screens (3a–u) the aldehyde was PAA (1a). All product nomenclature and m/z values assigned for each product are provided in Fig. apo-ObiD were pantetheinylated at room temperature for 2 h by combining 180 μM CoASH, 5 mM thiol, 10 mM MgCl2, 1 μM Sfp, 25 μM apo-ObiD or ObiF1 variant, and 25 mM sodium phosphate buffer, pH 7.5. For all reactions, ObiH at 5 μM was first incubated at room temperature with 500 μM of the aldehyde substrate along with 1 mM l-Thr, 500 μM thiol, and 5 μM PLP in 25 mM sodium phosphate buffer, pH 7.5 . After 1 h, 1 μM holo-ObiF1, 1 µM ObiF2, 1 μM holo-ObiD, 5 mM ATP, and 500 μM benzoic acid  were added to the ObiH reaction giving a final working volume of 300 μL for each experiment. For WT, del1304, rbs1303/4, and A841E additional experiments were performed with added MLP domain at a final concentration of 1, 10, or 25 μM. Reactions were allowed to proceed at room temperature and 100 μL aliquots for LCMS analysis were quenched at 30 min, 3 h, and 24 h time points by addition of 1 M HCl to give a final pH ~2. LCMS samples were spiked with 100 μM phenylalanine  as an internal standard and frozen at −80 °C. At the time of LC-MS analysis, the sample was thawed, centrifuged at 16,000 r.p.m. for 1 min to pellet solids, and analyzed by LCMS  water and (B) acetonitrile; method: 5% B to 100% B over 20 min; software: G2710 ChemStation). All experiments with ObiF1 mutant comparison and MLP supplementation were performed in at least duplicate as independent trials. All experiments with ObiF1 substrate screens were performed as a single trial. Extracted ion counts for each product were plotted either as bar graphs  or heat maps (ObiF1 substrate screens) using GraphPad Prism v7. Product assignments were confirmed by high-resolution mass spectrometry collected using an LTW-Velos Pro Orbitrap at the Donald Danforth Plant Science Center, St. Louis, MO. Note: PAA used for WT ObiF1 and D1254A, H1284A, D1254A/H1284A, C1146S, C1146A, D1177A, R1263A, R1190A, G1173D/D1254A, and G1173L was purchased from MilliporeSigma . During the course of revisions, PAA went on back order from the supplier. Therefore, PAA used for the WT ObiF1 and del1304, rbs1303/4, and A841E assays was synthesized from phenethyl alcohol using Dess-Martin periodinane oxidation in anhydrous CH2Cl2 followed by purification via silica gel column chromatography  to give PAA in > 95% purity with NMR characterization data matching authentic material from MilliporeSigma.For ObiF1 mutant assays the thiol trapping agent was GSH, the aldehyde substrate was PAA (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data


Synaptic dysfunction and synapse loss are prominent features in Alzheimer’s disease. Members of the Rho-family of guanosine triphosphatases, specifically RhoA, and the synaptic protein Arc are implicated in these pathogenic processes. They share a common regulatory molecule, the E3 ligase Ube3A/E6-AP. Here, we show that Ube3A is reduced in an Alzheimer’s disease mouse model, Tg2576 mouse, which overexpresses human APP695 carrying the Swedish mutation, and accumulates Aβ in the brain. Depletion of Ube3A precedes the age-dependent behavioral deficits and loss of dendritic spines in these mice, and results from a decrease in solubility following phosphorylation by c-Abl, after Aβ exposure. Loss of Ube3A triggers the accumulation of Arc and Ephexin-5, driving internalization of GluR1, and activation of RhoA, respectively, culminating in pruning of synapses, which is blocked by restoring Ube3A. Taken together, our results place Ube3A as a critical player in Alzheimer’s disease pathogenesis, and as a potential therapeutic target. Markel Olabarria et al. show that the loss of Ube3A plays a critical role in synaptic dysfunction, using an Alzheimer’s disease mouse model. This study establishes a molecular pathway that maintains a synaptic function and suggests Ube3A as a potential therapeutic target for Alzheimer’s disease. Mounting evidence suggests that impairment of cognitive abilities typically seen in the earliest clinical phases are due to prominent synaptic alterations and synapse loss, particularly in the entorhinal cortex (EC) and the hippocampus3, the principal areas affected in AD, and not primarily due to neuronal death. While the precise molecular mechanism remains unclear, it is widely accepted that AD-associated synaptopathy is caused by elevated levels of soluble oligomeric β-amyloid (Aβ), which specifically targets synapses and disrupts various signaling molecules and pathways involved in synaptic function5. A number of candidate molecules have been linked to AD-associated synaptic dysfunction.Alzheimer’s disease (AD), while classified as a neurodegenerative disease, is, at its core, a disease of synapses6, RhoA, Rac1 and Cdc42, which regulate synapses, more specifically dendritic spine morphology and function7, by regulating the actin cytoskeleton, the main structural component of dendritic spines. RhoA favors the destabilization of dendritic spines, while Rac1 and Cdc42 promote their stabilization and maturation8. Given their critical role in synaptic function, aberrant Rho-GTPase signaling leads to widespread neuronal network dysfunction and has been proposed to play a key role in AD. RhoA subcellular mislocalization and altered levels have been reported in both human AD brains and the human amyloid precursor protein (hAPP) Tg2576 (Swedish mutation) AD mouse model10. Furthermore, we recently demonstrated that synaptic dysfunction and synapse loss in cultured hippocampal neurons exposed to soluble oligomeric Aβ, and in the J20 hAPP (Swedish and Indiana mutations) AD mouse model11, are both preceded and dependent on increased RhoA activity12.Of particular interest are members of the Rho-family of guanosine triphosphatases (GTPases), a subfamily of the Ras superfamily of GTPases13. Arc plays a critical role in synaptic plasticity and memory formation by regulating postsynaptic trafficking of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors at excitatory synapses during long-term potentiation (LTP) consolidation and long-term depression evocation15. Several lines of evidence have shown Arc levels to be altered in human AD brains, in various AD mouse models, and in cultured hippocampal neurons exposed to oligomeric Aβ17.Another important molecule implicated in AD-associated synaptic dysfunction is the activity-regulated immediate-early gene Arc/Arg3.118. Ube3A is best known for its causative role in the rare neurodevelopmental disorder, Angelman Syndrome (AS). AS is characterized by microcephaly, severe intellectual deficits, abnormal sleep patterns, and hyperactivity, among several others, due to the loss of function of the imprinted UBE3A gene, located on chromosome 15q11.2-q1319. Several potential Ube3A substrates have been identified20, including ECT2, p53, p27, HR23A, Blk, and interestingly Arc and the RhoA-specific nuclear guanine nucleotide exchange factor (GEF) Ephexin-5, also known as ARHGEF15. One interesting characteristic observed in AS mouse models includes a significant reduction of dendritic spine density and length in neurons of the hippocampus and cortex21, akin to AD mouse models. This similarity led to us to investigate whether Ube3A dysfunction was also observed in the Tg2576 AD mouse model.How these two critical signaling molecules are dysregulated is currently unknown. However, they share one common regulatory molecule, the ubiquitin-protein ligase E3A, Ube3A/E6-AP22. The reduction of Ube3A protein leads to the accumulation of two of its targets, the synaptotoxic proteins Arc and Ephexin-5, leading to a decrease in surface expression of AMPA receptor subunit GluR1 and an increase in active RhoA, respectively. This culminates in altered synaptic function, and the aberrant pruning of dendritic spines and synapses. Finally, we show that restoring the levels of Ube3A protein in neurons completely protects against the synaptotoxic effects of Aβ oligomers, including the loss of surface-expressed GluR1, and the loss of dendritic spines. Taken together, these data suggest that the loss of Ube3A function plays a critical role in AD pathogenesis, specifically in synaptic dysfunction. Moreover, this study highlights Ube3A as a potential therapeutic target for AD.We found that the levels of Ube3A protein are reduced in the Tg2576 mice in an age-dependent manner, concomitant with a loss of dendritic spine density, and behavioral deficits. Moreover, we show that the decrease in Ube3A protein is neuron specific, and is triggered by elevated Aβ oligomers, which inactivate and decrease the half-life of the Ube3A protein through activation of the non-receptor tyrosine kinase, c-Abl9 overexpresses hAPP carrying the Swedish mutation (KM670/671NL), linked to early-onset familial AD. As previously shown23, we found that the Tg2576 mice developed amyloid plaques in the hippocampus  in Tg2576 compared to WT mice  family of receptor tyrosine kinases, EphB2. This results in ubiquitination and proteasomal targeting of Ephexin-5, catalyzed by Ube3Aage Fig. , we founage Fig. . Depletiage Fig. , and wasage Fig. . We alsoage Fig. . Togethe1–42 synaptotoxicity. Enriched primary rat hippocampal neurons, cultured for 21 days in vitro , transcribed as part of a large non-coding antisense transcript30. We first determined whether oAβ affected the levels of Ube3A messenger RNA (mRNA). However, quantitative real-time PCR (qPCR) analysis of oAβ-treated hippocampal neurons for 24 h did not show any differences in the levels of Ube3A mRNA -tagged Ube3A for immunoprecipitation. Consistent with our hypothesis, we found that treatment of these cells with either the c-Abl activator, DPH36, used as a positive control  chase assay Fig. . HoweverAs we observed in the Tg2576 mice hippocampi, the levels of Ube3A inversely correlated with the levels of Arc and Ephexin-5. To determine whether this correlation was also observed in hippocampal neurons, they were treated with Aβ for 8–10 h, and whole-cell lysates were collected for western blot analysis. This revealed an increase in the levels of both Ephexin-5 and Arc protein Fig. . Intrigu38. Aberrant upregulation of Arc has been shown to induce a decrease in surface expression of the AMPA receptor subunit GluR139. As previous studies have shown, when hippocampal neurons were treated with Aβ, inactivation of Ube3A correlated with a decrease in the levels of surface-expressed GluR1 subunit, as determined by western blot analysis of isolated biotinylated surface GluR1 . Once TMP is added, it binds to DD and stabilizes the fused protein, allowing it to accumulate in the cell. In our system, infection of hippocampal neurons with a DD-Ube3A-expressing lentivirus resulted in undetectable expression of exogenous Ube3A in the absence of TMP. However, the addition of TMP to the culture medium resulted in a dose-dependent increase in DD-Ube3A after 24 h , derived from the To test whether upregulation of Ube3A could rescue the effects of oAβ, cultured hippocampal neurons at 14 DIV were infected with DD-Ube3A-expressing lentivirus for 5 days, followed by TMP treatment for 48 h. The neurons were then treated with oAβ for 24 h, and whole-cell lysates prepared for western blot analysis or fixed for microscopy Fig. . Upregul26. The exact mechanism(s) leading to the upregulation of these two proteins is not fully understood. However, several prior studies suggest that oligomeric Aβ may be at least partially responsible, as it can induce their rapid expression in neurons44.While widespread synaptic dysfunction and the loss of synapses are invariable features of AD, the exact mechanism(s) driving these changes remains unclear. Accumulating evidence suggests that aberrant signaling by two key synaptic proteins may play an important role in AD-associated synaptopathy, namely Arc/Arg3.1 and Ephexin-5, whose levels are anomalously upregulated in the brain of AD patients, and in various AD mouse models45, is downregulated in cognitively impaired AD transgenic mouse brains, and in cultured primary rat hippocampal neurons treated with oligomeric Aβ, suggesting that Ube3A may also be a key player in AD-associated synaptic dysfunction. Consistent with this idea, the observed depletion of Ube3A paralleled the oAβ-induced increase in both Arc and Ephexin-5. Their accumulation can lead to decreased surface-expressed GluR1 and to the loss of dendritic spine density, respectively39. More importantly, restoration of Ube3A in oAβ-treated neurons was sufficient to completely block the induction of Arc and Ephexin-5, while also abrogating the loss of surface-expressed GluR1 and dendritic spine density, confirming the contribution of Ube3A to these processes. Interestingly, our data indicate that the decrease in Ube3A in response to oAβ exposure appears to be specific to neurons, as Ube3A levels were conversely increased in cultured primary rat astrocytes, suggesting a differential response to oAβ between neurons and astrocytes. Indeed, prior studies have shown that astrocytes, unlike neurons, are resistant to oAβ cytotoxicity, possibly due to the activation of distinct signaling pathways47.Herein we show that degradation of both Arc and Ephexin-5 may also be impaired in AD, perhaps contributing to their sustained expression in neurons exposed to oligomeric Aβ. In this report we show that the AS-associated protein, Ube3A, a key E3 ligase regulating the degradation of Arc and Ephexin-549. Our results show that activation of c-Abl by oAβ, or the c-Abl activator DPH, triggers the phosphorylation and inactivation of Ube3A, resulting in an unstable form of the protein, thereby reducing its half-life from over 24 h to about 8 h, with no effects on its transcription. Treating the neurons with the c-Abl-specific inhibitor, STI-571, completely blocked the phosphorylation and degradation of Ube3A. Interestingly, consistent with these results, this inhibitor has previously been shown to completely block the oAβ-induced loss of dendritic spine density and impaired LTP in vitro in cultured neurons34, and prevent tau phosphorylation in vivo and behavioral impairments in an AD mouse model50. Taken together, we propose a model 61. As such, there is tremendous interest in identifying the direct and indirect substrates of Ube3A20. This is particularly important for AD research, not only in light of the results of the studies herein, but due to the fact the Ube3A may also influence other aspects of AD pathogenesis, particularly APP and Aβ production. There is solid evidence for this idea. First, AS patients have significantly elevated plasma Aβ40 and 42 compared to age-matched control individuals62. Second, aberrant APP regulation has been reported in models of chromosome 15q11–13 duplication, in which Ube3A gene expression is increased 1.5- to 2-fold, which demonstrated that APP levels were decreased 10-fold relative to control samples63. Third, post-mortem analysis in 15q11–13 duplication (associated with ASD) shows that neurons from control patients showed higher deposition rates than neurons from subjects with ASD64. Fourth, Arc itself has been suggested as a key regulator of APP and BACE1 trafficking and Aβ production16. Taken together, these data suggest that Ube3A dysfunction may be intricately linked to AD pathogenesis.The consequences of loss of function of Ube3A have been well documented in the context of Angelman syndrome65, potentially resulting in numerous indirect substrates. In fact, while the expression of Arc does change in response to Ube3A, it may be an indirect substrate of Ube3A, highlighted by fact that varying results have been obtained by different groups regarding Arc as a direct Ube3A substrate67. Even previously well-established substrates, such as p53, have come into question, due to the fact that Ube3A does not ubiquitinate p53, at least in vitro54, and to the fact that in our studies p53 levels remained unaltered after Ube3A depletion. Nevertheless, the phenotypic similarities between AS and other neurodevelopmental disorders, and now with AD, can probably be explained by the significant overlap in downstream signaling pathways, which suggest that therapeutic strategies developed for one disorder may also be applicable to others, especially to correct like-phenotypes. More importantly, our results squarely place Ube3A as a prime target for future AD therapeutics.However, compiling a comprehensive list has not been without its share of difficulties and conflicting results. A recent study has demonstrated that disruption of Ube3A function may have widespread inhibitory effects on proteolytic activity of the proteasomed-lysine (PDL) hydrobromide mol wt > 300,000 (Sigma Aldrich), Dubelcco’s modified Eagle’s medium (DMEM) high glucose (ThermoFisher Scientific), DMEM/F12 (ThermoFisher Scientific), fetal bovine serum , Tris(2-carboxyethyl)phosphine hydrochloride , Complete Mini protease inhibitor cocktail (Roche Applied Science), Halt protease and phosphatase inhibitor (ThermoFisher Scientific), TRIzol reagent (Ambion), anhydrous dimethyl sulfoxide , methylene chloride (Sigma Aldrich), hexafluoro-2-propanol , 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate , phosphate-buffered saline pH 7.4 , tris-buffered saline pH 7.4 , Superblock (TBS) blocking buffer (ThermoFisher Scientific), paraformaldehyde 16% , Triton X-100 (ThermoFisher Scientific), radioimmunoprecipitation assay (RIPA) buffer (ThermoFisher Scientific), mammalian protein extraction reagent , and DNase/RNase-free water (ThermoFisher Scientific). Extraction buffer (EB) recipe: 20 mM [(4-[2-hydroxyethyl]−1-piperazineethanesulfonic acid]) (HEPES)], pH 7.4, 100 mM NaCl, 20 mM NaF, 1 % Triton X-100, 1 mM sodium orthovanadate, 5 mM EDTA.All chemicals used were of the highest grade available. Antibodies used in this study include the following  were prepared following a slightly modified version of the method of Brewer et al.40. Briefly, 20 nmole of siRNA was dissolved in 25 μl DNase/RNase-free water with 1.1 μl TCEP (17.6 mM) and incubated at room temperature for 1 h. Then, 25 μl of Penetratin-1 (dissolved in DNase/RNase-free water to 2 mg/ml) was added to the mixture with an additional 180 μl DNase/RNase-free water, and incubated for 15 min at 65 °C for 15 min, and then for 1 h at 37 °C. To visualize nucleic acid, 2 μl of the mixture was separated on 20% TBE gel (ThermoFisher Scientific) and stained with SYBR gold  for 15 min at room temperature.Oligonucleotide sequences were custom-ordered (Dharmacon) with a protected thiol functionality at the 5′-end . Oligonucleotide stocks were solubilized in DNase/RNase-free water at a 10 mM concentration. The delivery peptide Penetratin-1  was cross-linked via a Cys–Cys bond to the desired oligonucleotide as previously described9 used in this study was originally generated by Karen Hsiao and overexpresses a mutant form of APP (isoform 695) with the Swedish mutation (KM670/671NL). Male mice were kept under standard housing conditions (12 h light/dark cycle) with access to food and water. Age-matched WT littermates were used as controls. All experiments involving animals were performed under the guidelines of the Columbia University Institutional Animal Care and Use Committee.The Tg2576 mouse modelMice were trained to find a hidden platform in a circular pool of 1.10 m diameter located in a room with extra maze cues. The location of the platform (14 mm diameter) was constant for each mouse during training and was placed 1 cm beneath the surface of the water, maintained at 24 °C throughout the duration of the test. At the end of test, the mice were dried off and placed in a clean cage with extra paper towels to prevent hypothermia. Animals were then monitored inside their cage until eating, drinking, and ambulating normally.Mice were trained in sessions consisting of four trials a day and for seven consecutive days. Mice started from different quadrants on a random basis during each trial and all four quadrants were used on any given day. The maximum swimming time allowed on each trial was 60 s; if the mouse failed to reach the platform within 60 s, it was manually guided to the platform and kept there for 15 s.Probe trials  were conducted 24 h after the last training trial. During the probe test the platform was removed and mice were free to swim in the pool for 60 s. Time spent in each quadrant was measured. Trial and probe test sessions were recorded and analyzed with a video tracking system (Noldus). After the probe test, mice were tested for their ability to locate a visible platform in order to exclude differences in vision, swim speed, and motivation. These were performed for two consecutive days, 2 sessions/day with 3 trials/session . The times to locate the visible platform and the swim speeds were collected.Lyophilized oAβ (rPeptide) was dissolved to 1 mM in HFIP for 1 h in a tissue culture hood. The dissolved peptide solution was then aliquoted into Teflon-coated tubes and HFIP was allowed to evaporate at room temperature overnight under sterile conditions. The resulting pellet was further dried in a SpeedVac for 1 h at room temperature. Tubes were sealed and stored at −30 °C freezer with desiccator pellets. To prepare oligomeric oAβ, the pellet was resuspended in DMSO at 5 mM and sonicated for 15 min at room temperature. The solution was further diluted in DMEM/F12 to 100 μM and allowed to oligomerize at 4 °C for 24 h.69. Briefly, a slurry of tungsten particles  was prepared with DiI stain (5.0 mg) dissolved in methylene chloride, spread on a glass cover slide, and allowed to dry for 15 min. Dye-coated particles were resuspended in water (10 ml) and passed through a Tefzel tube (Bio-Rad) coated with 10 mg/ml solution of polyvinylpyrrlidone and allowed to dry for 1 h at room temperature. Particles were delivered into non-permeabilized PFA-fixed cells using the Helios gene gun system (Bio-Rad), the dye was allowed to diffuse overnight, and neurons were mounted onto glass slides using Gel Mount (Biomeda) for visualization.Cells were fixed in 4% PFA in PBS for 10 min at room temperature. Permeabilization was performed using 0.4% Triton X-100 in PBS for 20 min. Then, cells were incubated with the appropriate primary antibody in Superblock with 0.1% Triton X-100 for 12 h at 4 °C, followed by incubation in goat anti-mouse Alexa 568 in Superblock with 0.1% Triton X-100 for 1 h at room temperature. The protocol for diolistic labeling of neurons was adapted from Gan et al.Labeled neurons were imaged using the Laser Scanning Microscope 510 Meta confocal microscope (Zeiss), equipped with 40× 1.3 NA and 100× 1.4 NA oil-immersion objectives. The NIH Image software program ImageJ was used the quantify DiI-labeled neurons. An average of 12 compressed images (20 µm thick) consisting of pyramidal neurons were quantified for each treatment. Spines were counted with NeuronStudio software in a blinded manner and spine density was represented in number of spines per μm of dendrite length.Immunoprecipitations were performed using the Dynabeads Protein G IP kit (ThermoFisher Scientific). Briefly, cells were homogenized and incubated overnight at 4 °C with 1.0 μg of HA antibody or mouse IgG as a negative control. Immunocomplexes were captured by incubating with the magnetic beads on a rotator for 2 h at 4 °C and resuspended in cell lysis buffer with LDS-sample buffer for western blotting analysis.Whole frozen mouse hippocampi, human brain specimens, and cultured cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail using a Teflon dounce homogenizer and centrifuged at 14,000 rpm for 10 min at 4 °C. Supernatants were collected, normalized using a BCA assay kit (ThermoFisher Scientific), and prepared for loading using 4× sample buffer (ThermoFisher Scientific) and dithiothreitol 10× (ThermoFisher Scientific).70. Briefly, frozen brains were homogenized in a dounce homogenizer in 10 volumes (wt/vol) of EB buffer supplemented with protease inhibitor cocktail and 2 mM phenylmethane sulfonyl fluoride. Resulting homogenates were centrifuged  and the supernatants collected for total protein analysis. For sarkosyl-soluble and -insoluble fractions, homogenates were centrifuged  and the pellet was resuspended in sucrose buffer  and centrifuged again . The resulting supernatants were incubated on a rotator in 1% sarkosyl buffer for 1 h at 25 °C and centrifuged  to collect the pellet containing the insoluble proteins.Insoluble Ube3A protein extracts were prepared from frozen mouse brains as previously describedRNA from neurons was extracted using TRIzol following the manufacturer’s recommended protocol. Concentration and purity were assessed by measuring the O.D. at 260 and 280 nm with a NanoDrop (ThermoFisher Scientific) and 1 μg was used to synthesize complementary DNA (cDNA) using a first-strand cDNA synthesis kit (Origene) following the manufacturer’s protocol. qPCR was performed using the FastStart SYBR Green Master Mix (Roche Applied Science) on an Eppendorf Realplex Mastercyler using the following cycle parameters: 1 cycle at 95 °C, 10 min; 95 °C, 15 s, 58–60 °C, 30–60 s; 72 °C, 30–60 s; 40 cycles of amplification. Ube3A mRNA was quantified and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as a fold induction compared to control conditions. Primer pairs for Ube3A: F_5′-CGAGGACAGATCACCAGGAG-3′ and R_5′-TCATTCGTGCAGGCCTCATT-3′.g) for 5 min. Then, 30 μl was used for input  and the remaining lysates were incubated with Rhotekin-RBD (RhoA) protein beads (25 μl) by rotating for 1 h at 4 °C. Beads were washed 3 times with ice-cold PBS (500 μl) and collected, and bound proteins (active RhoA)  were eluted with 1× sample buffer (30 μl). Input and eluted samples were then analyzed by gel electrophoresis and western blotting using either a RhoA-specific antibody.Rho-GTPase pull-down assays were performed following the manufacturer’s protocol . Briefly, cells were washed once with ice-cold PBS and lysed with RIPA/M-PER buffer . Lysates were incubated on ice for 5 min, and centrifuged , where appropriate, with a significance threshold of p < 0.05.All statistical analyses were performed using GraphPad Prism software. Statistical significance was determined by Student's Further information on experimental design is available in the Description of Supplementary DataSupplementary InformationSupplementary Data 1Reporting Summary


Bioindicator species are extensively used for rapid assessment of ecological changes. Their use commonly focuses on changes in population abundance and individual sizes in response to environmental change. These numerical and demographic shifts likely have behavioral and physiological mechanistic drivers that, if understood, could provide additional insights into the use of these species as bioindicators of habitat health.Ocypode quadrata, is a global bioindicator species of human disturbance on sandy shores. Individual size and population abundance of O. quadrata decline dramatically at sites with human disturbance, and the causes of this phenomenon remain unclear.The Atlantic ghost crab, O. quadrata changes because of human disturbance. We also examine energy required for burrowing by O. quadrata across different levels of human disturbance.Here, we test the hypothesis that individual and population‐level changes at disturbed sites reflect changes in burrowing behavior and energetics. Specifically, we examine whether or not the burrowing behavior  of O. quadrata have the highest burrow fidelity and longevity at sites with low level of human impact, and weakest burrow fidelity and longevity at pristine sites. O. quadrata reduce the burrowing energy allocation by manipulating the burrow dimension and increasing the burrow longevity even under low levels of human disturbance.We show that Overall, this study shows that human disturbances not only change the behavior of organisms, but also shift energetic balance. Our results support the use of a bioenergetic approach to better understand how human disturbances influence natural populations, and the specific use of this approach with this bioindicator species. Human disturbance alter the behavior and therefore shift the energetic balance of the organisms. We predicted that O. quadrata will be forced to burrow more frequently on disturbed sites because their burrows would be destroyed by human activities more often compared with pristine sites. We further predicted that burrowing would represent an energetically expensive behavior and that this energetic cost should be influenced by changes in the frequency of burrows and the size of burrows across beaches with different levels of human disturbance.The most widespread responses of ghost crabs to human disturbances on sandy shores are decreases in population abundance and individual body sizes  of the Atlantic ghost crab, 17 Table . Specifi17 Table . We then2.2We used a mark–recapture study to examine burrow fidelity (described below). Prior to this, we used a preliminary study to determine whether our mark and recapture technique would likely alter burrow fidelity and longevity of ghost crabs. This preliminary experiment did not determine whether our procedure influenced the burrow fidelity of crabs. We therefore make the assumption that any impacts of our procedures on burrow fidelity did not differ across levels of human impact. We randomly selected one site for each of the four human disturbance levels. At each site, we selected newly created burrows in the upper zone  and designated them as control or treatment burrows. Burrow was identified as “newly‐created” by initially marking existing burrows and selecting new burrows that appeared beginning the next day for the study as they were created by crabs. We examined a total of 84 burrows  across the four sites. Each burrow was marked using a marking flag. We were forced to burry our marking flags in the sand at about a 20‐cm depth in the sites with human presence because beachgoers removed unburied flags. The experimental controls consisted of a set of burrows used by crabs that had not been marked. For these control burrows, we used a set of burrows that were newly created over a 3‐day period and examined these until they collapsed to determine the burrowing behavior and to measure burrow longevity. The experimental treatment consisted of a set of burrows used by crabs that had been marked for identification. For these treatment burrows, we set up simple traps (approximately 45 cm deep buckets buried flush with the sand and with a piece of rotting fish meat to lure crabs into the buckets) before sunset to capture the crabs that newly created their burrows. We also surrounded burrows and traps together using approximately 30 cm high plastic mesh to guide crabs into the traps. We were careful not to destroy the sand mounds and not to remove any material  from around the burrows, as individuals use sand mounds Hughes,  and mari2.3O. quadrata will enter any random burrow, we collected 10 individuals of various body sizes on Waties Island (one of our pristine sites). We kept these individuals in separate plastic containers for about 10 min, during which time we shook the containers gently to disturb them. We then chose random burrows that were matched to the size of individual crabs and released the individuals, one by one, next to the mouths of these burrows. We conducted this experiment at night when crabs are usually out of their burrows foraging, reducing the likelihood that these burrows were already occupied by their resident crab. When an individual did not immediately try to escape or enter the burrow, we pretended to catch them to elicit a response. We applied a Chi‐squared test to determine whether crabs entered random burrows. We only conducted this experiment at one of our pristine sites and therefore cannot be sure that the behavior does not differ with the level of human impact.We conducted a second preliminary experiment to determine whether crabs would use a burrow that they did not construct. If crabs will use a burrow that they did not construct, then they may avoid the energetic cost of burrow construction. During our study, we recognized that marked individuals returned to their own burrows immediately after we had released them. We also observed that foraging individuals run back to burrows from a certain distance  when they encounter humans. Thus, to test whether 2.4r2 = 0.98, Wolcott, To understand whether the number of days during which an individual uses the same burrow (burrow fidelity) varies based on the level of human disturbances, we applied a mark and recapture technique as described above at one site for each disturbance level. For the burrow fidelity, we sampled the newly created burrows over a 1‐week period in three replicate rectangular quadrats (10 × 5 m) situated on the seaward side of the dune vegetation at each site. We established our quadrats near the dune vegetation because the persistence of ghost crab burrows is directly affected by the tides  in three trial rectangular quadrats (20 × 10 m) in each site listed in Table To examine whether the persistence of the ghost crab burrows themselves was influenced by the level of human impact, we observed the longevity of ats 20 × 0 m in eaGeomorphological characteristics of sandy beaches and burrow densities at each site were determined as possible explanatory variables of the burrowing behavior  with ~5 cm moist sand. We weighed them and kept them without food for 3 days to standardize their hunger level, after which we fed them every other day with commercially purchased salmon for next 10 days. Each individual was offered 10% of its wet body weight at each feeding, and uneaten food was removed after 24 hr. All of this was done in an attempt to standardize the energetic conditions across crabs.We conducted an experiment to determine the energetic coast of digging for Following this holding period, we transported the crabs back to the Isle of Palm where they were collected. We created experimental chambers by excavating 20 pits in the sand at Isle of Palm on the beach berm. This was done at night to avoid changing the temperature of sand at depth via direct sunlight. Into each pit, we placed a 121 L plastic can  with approx. 50 small holes drilled into the sides and the bottom to allow moisture and temperature exchange between the inside of the chamber and the surrounding sediment. We then replaced the sand that had been removed into these experimental chambers. To mimic the sand compaction of the surrounding beach, we excavated another identical pit and measured the sand compaction every ~20 cm during excavation using a pocket penetrometer. Then, as the chambers were filled with sand, we measured the compaction inside the chambers at each 20‐cm interval and pressed the sand as necessary to achieve the same compaction level as was observed in the surrounding sediment. We surrounded the mouth of the chambers with a vertical cardboard barrier to keep the crabs from escaping and then allowed 4 hr for moister and oxygen levels in the chambers to equilibrate. We then released an individual treatment crab onto the sand surface in each chamber and allowed them to excavate burrows. During this time, we held the other 20 control crabs in their transport containers under the same ambient environmental conditions with the treatment crabs in the field. After about 2 hr, all experimental crabs had excavated a burrow. To force the individuals to leave their burrows, we used a smoke fumigator  from each leg muscle removed from the experimental and control crabs. Finally, we measured absorbance of our samples under a spectrophotometer (Biotek Synergy H1 Hybrid Reader working with software Gen5). The glycogen content of the samples was calculated from a standard curve [glycogen content (μg/mg of muscle tissue) = 1.9837 × OD(570 nm) − 0.0269; Rg Lucas, . Our visg Lucas, . SpecifiO. quadrata. We did this by combining our data on burrow longevity at each of our sites and the energy expended during the removal of 1 cm3 of sand , with overall burrow volume that we have previously measured at these same sites . Thus, we conclude that any artifact introduced by our capture and marking procedures was minimal.The trapping and marking technique used here to determine the burrow fidelity did not seem to alter the longevity of the burrows. The burrow longevity in the treatment group was slightly lower than in the control group under each level of human impact; however, none of these differences were significant . Specifically, we observed that 9 out of 10 individuals did not enter the foreign burrow that they were released next to, even though that burrow was large enough for them. Only one individual entered shallowly into the foreign burrow, staying very close to the entrance, and when we disturbed it using a thin stick, it left the burrow immediately and ran away rather than receding further into the burrow.We did not find any instances of a foreign crab occupying one of our focal burrows during any part of our study. This is also consistent with 3.3O. quadrata was significantly lower in pristine sites  compared with burrow fidelity in moderately impacted sites , highly impacted sites by people , and highly impacted sites by people and vehicles  and sand compaction . No impact of sand grain size on burrow fidelity was detected . No significant impact of the interaction terms was detected.The average burrow fidelity of 3.4−1, range = 1–6 day−1, n = 340) and in highly impacted sites by people and vehicles  was similar . Contrary to this, a greater burrow longevity was observed in moderately impacted sites  and in highly impacted sites by people  compared with pristine sites .Burrow longevity in pristine sites , between burrow density and sand compaction rate  and between sand compaction rate and the sand grain size . These interactions influenced burrow longevity . The glycogen content in the digging leg tissues of the individuals in the treatment group increased with crab size by 0.123 ± 0.031 μg for every mm increase in carapace width of the crab  under the influence of various levels of human disturbances. We have also demonstrated that O. quadrata spend a much higher amount of energy in pristine sites for their burrowing behavior compared with the sites disturbed by people. We have also shown that larger individuals generally return to the same burrow for a longer period of time compared with smaller individuals. Since burrows protect crabs from predators, cannibals, and environmental influences such as strong winds, cold, and hot weather , mole crabs (Emerita spp.), and sandy beach coleoptera   suggests that burrow maintenance is considerable. Further research is needed to examine the relative costs of initial burrow creation and burrow maintenance, as well as seasonal variation since crabs will demand more energy during the reproduction period.4.2Our study has at least two broader implications for the population ecology of species used as bioindicators of human disturbances. First, in agreement with countless other studies, our study shows that species alter their behaviors and daily activities, and therefore their energetic balance, in the presence of human disturbance. Despite the fact that lower population density and smaller individual sizes are widely used as common responses of bioindicator species to human disturbances (Carignan & Villard, Second, the link between the levels of human disturbance and energy allocation suggests that this mechanistic link may improve the power to predict the impacts of human disturbances. While examining changes in abundance and individual body sizes within populations is a quick and cost‐effective technique for assessing the extent of human disturbance (Carignan & Villard, None declared.MRG and BDG conceived and designed the experiments. MRG performed the experiments. MRG and BDG analyzed the data and wrote the manuscript. Click here for additional data file.