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. 2020 Jul 8;15(7):e0235925. doi: 10.1371/journal.pone.0235925

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity

Steven A Beasley 1, Chloe E Kellum 1, Rachel J Orlomoski 1, Feston Idrizi 1, Donald E Spratt 1,*
Editor: Michael Massiah2
PMCID: PMC7343168  PMID: 32639967

Abstract

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.

Introduction

Angelman syndrome (AS) is a neuro-genetic disorder that effects 1 in 15,000 people characterized by symptoms such as developmental delay, speech impairment, intellectual disability, walking and balance disorders, and epilepsy [1–3]. Individuals also demonstrate a unique behavioral pattern that typically includes a happy demeanor, easily provoked laughter, short attention span, sleep disturbance, and an affinity for water [2, 4]. AS is caused by the loss of gene function of the maternal copy of UBE3A on chromosome 15. UBE3A is paternally imprinted in neurons resulting in expression of the maternal allele alone, whereas other tissues retain normal biallelic expression patterns [2, 5–8]. The Human Gene Mutation Database (HGMD) currently lists 161 different genetic mutations of UBE3A [9]. Of the genetic mechanisms that have been described as the cause of AS, an estimated 70–80% of cases contain deletions in the maternal chromosome 15q11-q13 [2, 3]. Another 10–20% of affected individuals harbor mutations in their maternally inherited UBE3A gene, 3–5% of cases are due to two paternal copies of the chromosome, and lastly 3–5% of affected individuals have the paternal imprint of the maternal chromosome leaving no functioning copy of the UBE3A gene [2, 3].

UBE3A codes for the E3 ubiquitin ligase E6-Associated Protein (E6AP). This protein is a member of the Homologous to E6AP Carboxy-Terminus (HECT) family of E3 ubiquitin ligases and plays an important role in the ubiquitylation-signaling pathway. Ubiquitylation is a post-translational modification of targeted proteins that initiates processes such as protein degradation, intracellular trafficking, and other signaling events [10, 11]. Ubiquitin is transferred onto a substrate through a cascade of three enzymes (E1, E2, E3), with the combinatorial effect of the approximately 40 E2 and over 600 E3 enzymes ultimately determining substrate specificity [12–14]. The fate of the ubiquitylated substrate is determined by the ubiquitin linkage chain type by the E2/E3 combination [10, 11, 15]. For example, K29 and K48 linkages target a substrate protein for proteasomal degradation, while K63 linkages are involved in DNA repair mechanisms and intracellular targeting [10, 11, 16]. The dysregulation of any of these components involved in the ubiquitylation process leads to a myriad of different diseases including various cancers, developmental disorders, and neurodevelopmental disorders including AS [8, 16–18].

E6AP was first identified as an E3 ubiquitin ligase through its ability to target the tumor suppressor protein p53 for degradation in conjunction with the human papilloma virus protein E6 [19, 20]. E6AP is a 100 kDa protein that catalyzes the covalent attachment of ubiquitin to its various substrates through the use of its C-terminal HECT domain (residues 518–875). The HECT domain consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe) connected by a flexible three-residue hinge [21]. A broad cleft at the interface of the two lobes contains the catalytic cysteine required for ubiquitylation [21]. E6AP selectively builds K48-polyubiquitination chains, consistent with its ability to target substrates for proteasomal degradation, and this ubiquitin chain-linkage specificity is located in the C-lobe of the E6AP HECT domain [22]. E6AP has been shown to interact with numerous cellular proteins and can regulate a number of different homeostatic cellular processes [23]. Prime examples of E6AP-regulated processes include cell cycle control through centrosomal regulation [24], DNA repair through its interaction with UV excision repair protein RAD23 homolog A (HHR23A) [25], targeting tuberin (TSC2) for proteosomal degradation [26], signal transduction by binding to multiple Src family tyrosine kinases [27], breast cell proliferation through calmodulin/Ca2+mediated proteosomal degradation of the estrogen receptor (ER) [28], and coordinating the inflammatory response in conjunction with annexin A1 [29].

While the genetic link between mutations in UBE3A and AS is well established in the literature [1–4, 6, 7], the effect that each specific AS mutation has on the translated protein product have not been fully characterized or well understood. In this study, we show that the AS I827K substitution (also described as I804K based on an alternate open reading frame start codon [30–33]) partially disrupts the overall 3D fold of the HECT C-terminal lobe of E6AP leading to its aggregation and diminished E6AP-ubiquitylation activity in vitro. We unambiguously demonstrate that the AS I827K mutation in UBE3A is a loss of function mutation. This biophysical study clarifies how the I827K substitution in the C-terminal lobe domain of E6AP contributes to the Angelman syndrome phenotype.

Methods and materials

Cloning and site-directed mutagenesis

The original DNA construct for the human HECT C-lobe of E6AP (E6APC-lobe; Uniprot Q05086, residues 761–875) was codon optimized and synthesized by ATUM (Newark, CA, USA) [34–36]. The gene was cloned into an ampicillin-resistant T7-inducible plasmid with an N-terminal His6 affinity tag followed by a TEV protease cleavage site (ENLYFQ/GS). The resulting His6-TEV-E6APC-lobe vector was subsequently used as the template to insert the I827K mutation using the SPRINP protocol [37]. The catalytic cysteine was changed to an alanine (C843A) using the same protocol. Due to subsequent precipitation issues of the I827K substituted construct and to increase solubility, the E6APC-lobe-I827K open reading frame was subcloned into an expression vector with an N-terminal His6-SUMO fusion tag using compatible 5’ BamHI and 3’ XhoI restriction sites. The HECT domain of E6AP (residues 518–875) was PCR amplified from a plasmid coding for the full-length E6AP purchased from Addgene (Plasmid #8655; Watertown, MA, USA) [27] and subcloned into the His6-SUMO vector using compatible 5’ BamHI and 3’ XhoI sites. All plasmids (pHis6-TEV-E6APC-lobe, pHis6-SUMO-E6APC-lobe-I827K, pHis6-SUMO-E6APHECT and pHis6-SUMO-E6APHECT-I827K) were isolated using the Monarch Plasmid Miniprep kit (New England Biolabs, Ipswich, MA, USA), quantified by A280 using a Nanodrop OneC UV-Vis spectrophotometer (Thermo-Fisher, Waltham, MA, USA), and verified by DNA sequencing (Macrogen, Cambridge, MA, USA).

Protein expression and purification

The pHis6-TEV-E6APC-lobe, pHis6-SUMO-E6APC-lobe-I827K, pHis6-SUMO-E6APHECT and pHis6-SUMO-E6APHECT-I827K expression plasmids were transformed into E. coli BL21 (DE3) RIL+ competent cells and grown at 37°C in Luria-Bertani media supplemented with ampicillin 100 mg/L and chloramphenicol 34 mg/L. The E6AP proteins grown for heteronuclear NMR analysis were grown in minimal M9 media (2 x 1L) supplemented with 1 g/L of 15NH4Cl and 2 g/L of 13C-glucose as the sole nitrogen and carbon sources. When the cultures reached an OD600 of 0.6–0.8, protein expression was induced with the addition of 0.5 mM IPTG for 20 hours at 16°C. The cells were harvested by centrifugation 6000 x g for 10 minutes at 4°C using a Sorvall LYNX 4000 superspeed centrifuge with a Fiberlite F10-4x1000 LEX Carbon Fiber rotor (Thermo-Fisher) and resuspended in cold wash buffer (50 mM Na2HPO4 pH 8.0, 300 mM NaCl, 10 mM imidazole) supplemented with ProBlock Gold Bacterial Protease inhibitor cocktail (GoldBio, St. Louis, MO, USA). The cells were then lysed using an Avestin EmulsiFlex-C5 Homogenizer (Avestin, Ottawa, ON, Canada) and clarified by ultracentrifugation using an Optima L-80 XP ultracentrifuge with a Ti 70.1 rotor (Beckman-Coulter) for 40 minutes at 41,000 rpm at 4°C. The clarified supernatant containing the desired His6-tagged E6AP protein was then isolated using 5 mL of HisPur Ni-NTA resin (Thermo-Fisher) and eluted with elution buffer (50 mM Na2HPO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). Fractions containing E6AP protein were pooled and incubated at 25°C for one hour in the presence of TEV or SUMO protease to cleave the N-terminal His6 or His6-SUMO tag, followed by overnight dialysis at 4°C against wash buffer to remove excess imidazole. To separate the cleaved His6- or His6-SUMO tag and His6-tagged protease from the desired E6AP protein, the cleaved sample was passed through the HisPur Ni-NTA resin column a second time and the flow-through containing the desired tag-free E6AP protein was collected. The E6AP protein was then concentrated using a 10 MWCO Amicon Ultra-15 Centrifugal Filter (Millipore) to about 1 mL, and run through a Superdex-75 Gel Filtration Column using an AKTA Pure 25L Fast Performance Liquid Chromatography (FPLC) system with gel filtration buffer (50 mM HEPES, 100 mM NaCl, 1 mM DTT, pH 7.5 at 4°C) at a flow rate of 1 mL/min. Due to inherent insolubility issues and after numerous attempts, the E6APHECT and E6APC-lobe-I827K proteins were unable to be passed through the size exclusion column.

Circular dichroism spectroscopy

Circular dichroism spectroscopy was performed on the E6APC-lobe and E6APC-lobe-I827K substituted protein using a JASCO J-815 CD spectropolarimeter. The proteins were prepared by buffer exchange into low salt (10 mM Na2HPO4 pH 7.4, 30 mM NaCl) using a 10 MWCO Slide-A-Lyzer Dialysis Cassette (Thermo-Fisher). The samples were diluted to 70 μM and loaded into a quartz cuvette with a 1 mm pathlength. Wavelength scans were averaged from six trials recorded from 260 to 195 nm at 10°C using 1 nm increments and an averaging time of 1 second. The same sample was used afterwards to obtain a melting curve of the mutant monitoring changes in α-helical content at 222 nm over a temperature range of 5–90°C, temperature slope 1°C/min, data pitch 0.3, response 4 seconds, bandwidth 1 nm, sensitivity standard 100 mdeg, and voltage of 600 mV.

Ubiquitin activity assays

Ubiquitination assays were conducted with 10 μM Alexa Fluor 647 N-terminally labeled ubiquitin, 10 μM E1 activating enzyme UBE1 (Uba1), 15 μM E2 conjugating enzyme UBE2L3 (UbcH7), and E3 ligase (35 μM E6APC-lobe or E6APHECT, as well as their variants), 2 μM DTT, 20 mM ATP, 40 mM MgCl2 in 50 mM HEPES pH 7.5, 100 mM NaCl. Each reaction was incubated at 37°C in a water bath for 30 minutes. To determine the presence of ubiquitin~thioester intermediates, appropriate samples were supplemented with 10 mM DTT. Reactions were terminated by adding gel loading dye and heating at 95°C in a dry bath for one minute. The samples were then loaded onto a Bis-Tris gel at pH 6.4 and run for 1 hour at 120 V. The gels were removed from the apparatus and immediately visualized on an iBright FL1000 imaging system (Thermo-Fisher) using the fluorescent gel imaging setting for Alexa Fluor 647.

Heteronuclear NMR spectroscopy

The 1H-15N heteronuclear single quantum correlation (HSQC) spectra [38] of 15N-labeled E6APC-lobe (3.5 mM) and E6APC-lobe I827K (188 μM) were collected at 25°C in a Varian Inova 600 MHz 4-channel solution-state NMR spectrometer equipped with a 5-mm PFG triple-resonance probe housed and maintained in the Carlson School of Chemistry and Biochemistry at Clark University. The samples were prepared to 600 μL in NMR buffer (20 mM Na2HPO4 pH 7.0, 100 mM NaCl, 2 mM TCEP, 1 mM EDTA), 10% D2O. The spectra were referenced to the methyl peaks of 2 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) set at 0 ppm and 2 mM imidazole was added as an internal pH indicator [39]. The backbone resonances were sequentially assigned using standard 2D and 3D experiments from the Varian Biopack including 1H-15N-HSQC, HNCACB [40], CBCA(CO)NH [41], HNCA [42–44], HN(CO)CA [44, 45], HN(CA)CO [46], and HNCO [42–44]. Side chain assignments were determined using C(CO)NH [47], H(CCO)NH [47, 48], as well as both aliphatic and aromatic [49], HCCH-TOCSY [50, 51] and 1H-15N and 1H-13C-NOESY experiments [52–54]. All data were processed using NMRPipe and NMRDraw [55] and the spectra were analyzed using NMRViewJ [56, 57]. All chemical shift assignments and experiments were deposited into the Biological Magnetic Resonance Databank (http://www.bmrb.wisc.edu) under accession code 50084.

Results and discussion

The I827K Angelman syndrome substitution destabilizes E6APC-lobe

Wild-type E6APC-lobe and E6APC-lobe-I827K Angelman syndrome substituted protein showed similar secondary structural content by circular dichroism, with minima at 208 and 222 indicating both proteins are predominantly α-helical at 10°C (Fig 1A). To test the thermal stability of wild-type E6APC-lobe and E6APC-lobe-I827K, melting curves were obtained for each protein from 5–90°C. The melting curve showed a marked decrease in thermal stability of the E6APC-lobe-I827K (Tm of 47.7°C) when compared to E6APC-lobe wild-type (Tm of 57.7°C) demonstrating that the Angelman syndrome I827K substitution appears to partially destabilize the 3D fold of the protein (Fig 1B). The Angelman mutant appears to start unfolding around 40°C, close to the physiological temperature of 37°C, whereas the E6APC-lobe wild-type begins to unfold around 55°C. These results indicate that the AS substitution has a similar global fold at cooler temperatures similar to the wild-type, whereas the melting curve suggests that the I827K AS substitution is detrimental to E6APC-lobe stability.

Fig 1. Circular dichroism spectra for E6APC-lobe (wild-type—○, I827K substitution—◆).

Fig 1

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A) Wavelength scans of the E6APC-lobe constructs show similar secondary structure content at 10°C. B) Melting curves show the E6APC-lobe-I827K substitution has a lower the Tm (47.7°C) than wild-type E6APC-lobe (57.7°C).

I827K Angelman syndrome substitution decreases E6AP ubiquitylation

The transfer of ubiquitin through the ubiquitylation pathway onto the catalytic cysteine of the E6AP was assessed using a fluorescent ubiquitin activity assay. Using this assay, we are able to observe the sequential transfer of fluorescent ubiquitin from the E1 activating enzyme UBE1 (aka Uba1), to the E2 conjugating enzyme UBE2L3 (aka UbcH7), and finally onto the HECT E3 ubiquitin ligase. This assay allowed for the direct comparison of wild-type to the I827K Angelman substituted E6APHECT and E6APC-lobe activities. As shown in Fig 2, the progression of the ubiquitylation cascade as ubiquitin is transferred sequentially onto the E1 activating enzyme UBE1 in an ATP-dependent manner, followed by a transfer onto the E2 conjugating enzyme UBE2L3. Furthermore, the labile nature of the thiolester UBE1~ubiquitin and UBE2L3~ubiquitin complexes was demonstrated by the addition of the reducing agent DTT (Fig 2). Polyubiquitin chains formation by wild-type E6APHECT decreased in the presence of DTT. It is noteworthy that the addition of DTT did not result in a complete reduction of the E3~ubiquitin thioester bond in the E6APHECT, as was expected, possibly due to non-specific autoubiquitylation of the E6APHECT during the reaction. This is consistent with previous reports of the isolated E6APHECT being able to autoubiquitylate itself in the absence of substrate [58, 59]. Interestingly, the I827K substitution on the other hand did not result in any polyubiquitin chain formation, demonstrating that the AS substitution decreases ubiquitylation activity. With regards to the E6APC-lobe, the AS I827K mutation also showed diminished activity compared to the wild-type E6APC-lobe. Polyubiquitin chains were not formed in either of the reactions, indicating that the N-lobe is required for the efficient catalysis of chain formation, consistent with activity assays for E6APC-lobe [59] and other isolated HECT E3 ubiquitin ligase C-lobes for HUWE1, Smurf2, and UBR5 [60, 61]. Interestingly, the AS I827K substituted E6APC-lobe was still able to be monoubiquitinated, albeit to a lesser extent that wild-type E6APC-lobe. This observation could possibly be due to the structural disruption of the I827K mutation, which prevents the proper presentation of the E6APC-lobe catalytic cysteine to E2~ubiquitin complex in the absence of the E6AP N-lobe. Mutating the catalytic cysteine to an alanine (C843A) resulted in the complete loss of ubiquitylation activity, as would be expected due to the requirement of E6AP C843 in thiolester bond formation with ubiquitin.

Fig 2. E6AP I827K is a loss of function substitution.

Fig 2

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The ubiquitylation activity assay of wildtype or I827K substituted E6APHECT and E6APC-lobe consisted of a 30-minute incubation at 37°C of various combinations of 10 μM ubiquitin N-terminally tagged with Alexa Fluor 647, 10 μM E1 ubiquitin activating enzyme (UBE1), 10 μM E2 ubiquitin conjugating enzyme (UBE2L3), and 35 μM E3 ubiquitin ligase in a buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 16 mM ATP, and 40 mM MgCl2. 10 mM DTT was added to alternate samples to reduce any thioester bonds present. The samples were run by electrophoresis a 15% Bis-Tris polyacrylamide gel and visualized on a ThermoFisher iBright FL1000 imaging system.

NMR structural analysis of E6APC-lobe

The 1H-15N heteronuclear single quantum correlation (HSQC) spectra for 15N-labeled wild-type E6APC-lobe and the E6APC-lobe-I827K substituted protein were used to analyze possible structural changes caused by the AS mutation. The wild type E6APC-lobe spectrum showed a well-folded protein, with well dispersed peaks, indicating that each amino acid was located in its own unique chemical environment (Fig 3A). In contrast, the spectrum of the AS I827K substituted E6APC-lobe showed many collapsed amide peaks that were mostly localized to the center of the spectrum (Fig 3B) indicating that the protein was partially unfolded. If the protein was completely unfolded protein would have peaks collapse to the center of the spectrum as solvent exposed peaks no longer experience shielding effects of neighboring amino acids. The disappearance of many peaks can be explained by the decreased signal/noise ratio due to the lower protein concentration as well as the line broadening effects caused by protein aggregation [62]. This is consistent with our inability to purify E6APC-lobe I827K protein after multiple attempts using gel filtration chromatography due to the protein aggregating and eluting in the void volume. Furthermore, the concentration used for I827K substituted protein in NMR was similar to the concentrations used in our activity assays and CD experiments, where we did not observe any protein precipitation issues.

Fig 3. The I827K substitution destabilizes the 3D fold of E6APC-lobe (residues 761–875).

Fig 3

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(A) The assigned 1H-15N-HSQC spectrum of 3.5 mM human E6AP catalytic C-lobe (residues 761–875) using the one-letter amino acid code and residue number according to the human E6AP sequence. The spectrum was determined using standard 3D heteronuclear experiments, and side chain amides for asparagine and glutamine are connected with a horizontal line. (B) The E6APC-lobe I827K sample was collected under identical conditions as the wild-type except it was only 188 μM and increased transients collected due to the inherent instability of the protein. The decreased signal intensity and partial collapse amide peaks represent the aggregation of the protein caused to the I827K residue substitution. The NMR samples contained 20 mM Na2HPO4 pH 7.0, 100 mM NaCl, 2 mM TCEP, 1 mM EDTA, and 10% D2O/90% H2O, with imidazole used as an internal pH standard and DSS as the reference point. All data were collected at 25°C on a Varian Inova 600-MHz NMR spectrometer.

Based on the known E6AP crystal structure (PDB 1D5F) [21], we hypothesize that the AS I827K substitution leads to a structural disruption of the E6APC-lobe due to a hydrophobic residue being switched to a residue with a positively charged sidechain terminus that would disrupt the hydrophobic network in the core of the domain. The I827 sidechain is not solvent accessible and would not readily accommodate the polar terminus of the lysine sidechain. This is corroborated by the NOESY data that shows the I827 amino acid side chain methyl groups make numerous NOE contacts to several different surrounding hydrophobic side chains including T774, Y776, I787, F790, W791, L824, and M825 (Fig 4). The same rationale would apply to the newly published structure of the domain-swapped E6AP dimer (PDB 6TGK), as I827 is still found embedded in the monomeric hydrophobic core and is not involved in the dimerization interface [63]. This structural disruption likely induces an allosteric change that reduces the catalytic activity and leads to its subsequent aggregation. This is supported by the CD data showing the presence secondary structure at 37°C as well as our observation of aggregated protein eluted in the void volume using gel filtration chromatography. The loss of structural integrity due to the AS I827K substitution correlates with the loss of ubiquitylation activity for E6AP (Fig 2). We are confident that our NMR resonance assignments for the E6APC-lobe (residues 761–875) are correct and complete as a chemical shift index analysis, which predicts the secondary structure elements based upon chemical shift deviations of the backbone atoms (Ca, C’, Cβ, N, Ha, and NH) [64], are in good agreement with the known tertiary structure of C-terminal lobe of E6AP (Fig 5).

Fig 4. The I827 residue is an integral residue in the hydrophobic core of E6APC-lobe.

Fig 4

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(A) The E6AP structure (PDB 1D5F) was rendered in Pymol highlighting the I827 residue (yellow with magenta methyls) surrounded by several aromatic and hydrophobic residues (cyan). (B) Representative 1H-1H-NOE strip plots for the I827 methyls showing numerous strong contacts with the surrounding hydrophobic atoms of core residues including T774, Y776, I787, F790, W791, L824, M825, and I827. The authenticity of the assignment was confirmed by reciprocal NOEs from the identified atoms.

Fig 5. Predicted secondary structural regions of E6APC-lobe.

Fig 5

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The probability plot was made by inputting the experimentally determined resonance assignments for E6APC-lobe into the online webserver CSI 3.0 [64]. The propensity to form an α-helix or β-strand are denoted in red and blue, respectively. The position of the I827 residue is marked with a star.

This study provides a structural and biophysical rationale for the E6AP ubiquitin ligase activity loss due to the I827K substitution in AS. This is in good agreement with a previous report that showed the AS I827K substitution resulted in decreased ubiquitylation of the E6AP substrate HHR23A and instability in vivo [31]. Continued studies on the E6AP structure and function will help to understand how AS genetic mutations in UBE3A result in enzymatic insufficiency and may lead to potential treatments to alleviate the Angelman Syndrome phenotype.

Supporting information

S1 Raw images

(PDF)

Click here for additional data file. (424.8KB, pdf)

Acknowledgments

The authors thank Dr. Guoxing Lin for maintaining the 600 MHz NMR spectrometer housed in the Carlson School of Chemistry and Biochemistry at Clark University.

Data Availability

All relevant data are within the manuscript. All chemical shift assignments and experiments were deposited into the Biological Magnetic Resonance Databank (http://www.bmrb.wisc.edu) under accession code 50084.

Funding Statement

This work was supported by the National Institutes of Health (R15GM126432 to D.E.S.; www.nigms.nih.gov) and start-up funds from Clark University (D.E.S.; www.clarku.edu). The funders did not play any role in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript.

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Decision Letter 0

Michael Massiah

24 Apr 2020

PONE-D-20-07946

An Angelman Syndrome Substitution in the HECT E3 Ubiquitin Ligase C-terminal Lobe of E6AP Affects Protein Stability and Activity

PLOS ONE

Dear Prof. Spratt,

Thank you for submitting your manuscript to PLOS ONE. First, I would sincerely like to apologize for the tardiness in getting this manuscript reviewed. The response to COVID19 has disrupted the schedule of reviewers and this certainly contributed to the slowness. In addition to having one outside reviewer, I also reviewed the manuscript to provide a timely decision for any revision. Below I state my review.

       I agree that I827K mutation has  structural effects on the protein but it is not clear from the HSQC that the C-lobe is completely unfolded. There are still very nicely dispersed signals and the low s/n may be due to both signal broadening due to aggregation and partial unfolding of the protein, and the low concentration of the protein. I wonder if a much longer HSQC of the mutant can be acquired to confirm unfolding. The structure shows that the Ile827 is not completely buried and could accommodate the K mutation. In figure 5, it may be more appropriate to show chemical shift Index rather than the probability plot to confirm that the wt C-lobe is the consistent with the predicted structure.

     In several instances, the concentration of the mutant C-lobe was indicated as 188 or 190 mM (millimolar). I presume you mean micromolar. Also is the concentration of the wt c-lobe indeed 3.5 mM, that seems very high for a domain that requires and make extensive contact with the N-lobe. Was the concentration measured with SUMO attached?

     Can you also provide a statement on whether HECT E3 ligase can poly-autoubiquitinates. Our experience with HECT is that they auto-monoubiquitinate. The fact that the wt- and mutant C-lobe are mono-ubiquitinated suggest that the two protein structures are fairly intact because the interaction with the E2 enzyme is necessary for the ubiquitination. It would also be worthwhile to note precedents that the C-lobe can be ubiquitinated by the E2 enzyme by itself.

With mine and that of the other reviewer, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Michael Massiah

Academic Editor

PLOS ONE

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Reviewer #1: The ubiqutination is one of the most important post-translational modification responsible for protein degradation. The E3A ligase, coded the E6AP protein belonged to HECT ligase family. An Angelman Syndrome observed due to mutation in C-terminal part of HECT domain (C-lobe). The I827K substitution one of the possible mutation leaded to decrease ubiquitin thioester formation and destroy the protein degradation pathway.

The analyzed construct E6AP(C-lobe) analyzed in manuscript comprises residues 761-875 (115 a.a. molecular mass 13 kDa), which is suitable for studies with CD and NMR spectroscopy. For the wild-type E6AP(C-lobe) authors provided high-quality experimental data including 3D 13C and 15N NOESY spectra (Figure 4), which can be used for evaluation of high-resolution 3D structure of the E6AP(C-lobe). However, experimental conditions deserve some criticism, first of all due to the pH of buffer used in experiments close to the isoelectric point (estimated pI 6.94).

As demonstrated by authors, the mutation I827K lead to structural changes, which are clearly visible with CD and NMR techniques. Nevertheless, I couldn’t exclude that observed data are artifact due to experimental conditions. The 15N HSQC spectrum for E6AP(C-lobe) I827K mutant (Figure 3B) suggests a strong aggregation phenomenon rather than unfolded protein. To discriminate between these hypotheses, the authors would have to perform additional experiments.

The short run of structural minimization after I827K substitution shows visible changes in orientation of side-chains of hydrophobic residues, but such dramatic differences were not confirmed experimentally. I could note that I827K mutation increases the pI up to 8.03, which is still very close to pH buffer used in experiments. Further, the K827 together with K829 evidently forms a hydrophilic cluster characterized by positive charge which can destabilize 3D structure of the E6AP(C-lobe). The computer simulations can help understanding that process in details. It’s not surprising that the mutation can facilitate allosteric effects on catalytic C843 resulting in decreased interaction with the polyubiquitination chain.

Summarizing. To discriminate between unfolded or molten globule state, there are some additional experiments required to extract the size of protein in solution, which would help understanding structural changes under I827K mutation. First, we suggest to measure translation diffusion with NMR spectroscopy (DOSY package). To my knowledge, Varian Inova 600 spectrometer is equipped with Performa IV z-gradient unit generated up to 70 G/cm. This is enough to measure diffusion for 13 kDa protein. On the other hand, other experiments can be used, such as the DLS measurements, to estimate size of particles in solution.

Minor points. Authors have to be more careful about manuscript preparation. The experimental conditions for the NMR experiments are different in ‘Materials and Methods’ and in Caption to Figure 3. Also the Figure 4A contains a wrong number of W827 (should be W791).

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PLoS One. 2020 Jul 8;15(7):e0235925. doi: 10.1371/journal.pone.0235925.r002

Author response to Decision Letter 0

8 Jun 2020

5 June 2020

Dr. Michael Massiah

Academic Editor, PLOS ONE

Dear Dr. Massiah,

Thank you for the opportunity to resubmit our revised manuscript titled “An Angelman Syndrome Substitution in the HECT E3 Ubiquitin Ligase C-terminal Lobe of E6AP Affect Protein Stability and Activity” that we originally submitted to PLOS ONE on March 19th, 2020. Please find our responses to yours and the reviewer’s specific comments below:

4 April 2020

PONE-D-20-07946

An Angelman Syndrome Substitution in the HECT E3 Ubiquitin Ligase C-terminal Lobe of E6AP Affects Protein Stability and Activity

PLOS ONE

Dear Prof. Spratt,

Thank you for submitting your manuscript to PLOS ONE. First, I would sincerely like to apologize for the tardiness in getting this manuscript reviewed. The response to COVID19 has disrupted the schedule of reviewers and this certainly contributed to the slowness. In addition to having one outside reviewer, I also reviewed the manuscript to provide a timely decision for any revision. Below I state my review.

Authors’ response: We thank the reviewer and you for reviewing our manuscript during these difficult times. Please excuse our delayed response to yours and the reviewer’s comments – Clark University operations were dramatically affected by the COVID-19 pandemic. We appreciate all of your helpful comments to improve our manuscript and we have addressed and/or incorporated many of your suggested edits.

I agree that I827K mutation has structural effects on the protein but it is not clear from the HSQC that the C-lobe is completely unfolded. There are still very nicely dispersed signals and the low s/n may be due to both signal broadening due to aggregation and partial unfolding of the protein, and the low concentration of the protein. I wonder if a much longer HSQC of the mutant can be acquired to confirm unfolding. The structure shows that the Ile827 is not completely buried and could accommodate the K mutation.

Author’s response: Thank you for this constructive feedback. Upon further examination of our data, we agree with you that the C-lobe is not completely unfolded. To address this, we have modified the language in the manuscript to state that the I827K Angelman Syndrome substitution causes the partial unfolding and aggregation of the protein. This is in good agreement with our CD data that shows the protein is still folded at temperatures up to the lowered Tm for the mutant. We also observed in our previous gel filtration elution profiles that a significant amount of the E6AP C-lobe I827K protein eluted in void volume indicating that the protein aggregated during purification. The original 1H-15N HSQC that we collected with 64 transients (8x longer than E6AP C-lobe wild-type) was due to significant line broadening that we can attribute to slower protein tumbling caused by protein aggregation. Using Pymol, we observe that the I827 residue is completely buried in the hydrophobic core and is not solvent accessible in the structure (PDB 1D5F), which is in good agreement with the NOE strips shown in Figure 4B. We do not think that the charged ε-amine of the lysine would be easily accommodated within the hydrophobic core, but it does result in the partial unfolding and aggregation of the E6AP C-lobe I827K protein.

In figure 5, it may be more appropriate to show chemical shift Index rather than the probability plot to confirm that the wt C-lobe is the consistent with the predicted structure.

Author’s response: Thank you for this suggestion. The software CSI 3.0 created by Dr. Wishart’s group uses the latest algorithms that incorporates the chemical shift index. We feel the probability plot is appropriate for this figure and would like to keep it as is. The predicted secondary structure boundaries in our CSI 3.0 plot are also in good agreement with the solved E6AP structure (PDB 1D5F) shown in Figure 4.

In several instances, the concentration of the mutant C-lobe was indicated as 188 or 190 mM (millimolar). I presume you mean micromolar.

Author’s response: Thank you for pointing out these typos. The concentration of the mutant C-lobe was indeed in micromolar (μM) and the corrections have been made in the manuscript. The discrepancy of 188 or 190 μM was due to a rounding decision made by different authors that worked on the manuscript. This value has been changed in the manuscript to 188 μM for consistency.

Also is the concentration of the wt c-lobe indeed 3.5 mM, that seems very high for a domain that requires and make extensive contact with the N-lobe. Was the concentration measured with SUMO attached?

Author’s response: The HECT C-lobe is distinct from the N-lobe in all structures of the HECT solved thus far, with the ability to pivot along a small tether. In our experience in working on some of the 28 human HECT constructs, but certainly not all, we have been able to achieve very high concentrations typically in the millimolar (mM) range. For example, in our recent publication on the ITCH C-lobe resonance assignments (Beasley et al., 2019) the 13C-15N enriched recombinant protein was concentrated to 2.6 mM. The His6- or His6-SUMO tag was removed from the constructs prior to NMR data collection and the protein concentrations were determined in the absence of the N-terminal affinity tag.

Can you also provide a statement on whether HECT E3 ligase can poly-autoubiquitinates. Our experience with HECT is that they auto-monoubiquitinate. The fact that the wt- and mutant C-lobe are mono-ubiquitinated suggest that the two protein structures are fairly intact because the interaction with the E2 enzyme is necessary for the ubiquitination. It would also be worthwhile to note precedents that the C-lobe can be ubiquitinated by the E2 enzyme by itself.

Author’s response: Thank you for this helpful suggestion. We have modified the text and expanded our discussion regarding this in our manuscript and included citations for previous reports of the E6AP HECT domain’s autoubiquityation activity. We have also included text referencing previous activity assays for the isolated HECT C-lobes of E6AP, HUWE1, Smurf2, and UBR5.

With mine and that of the other reviewer, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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Reviewer #1’s comments:

The ubiqutination is one of the most important post-translational modification responsible for protein degradation. The E3A ligase, coded the E6AP protein belonged to HECT ligase family. An Angelman Syndrome observed due to mutation in C-terminal part of HECT domain (C-lobe). The I827K substitution one of the possible mutation leaded to decrease ubiquitin thioester formation and destroy the protein degradation pathway.

The analyzed construct E6AP(C-lobe) analyzed in manuscript comprises residues 761-875 (115 a.a. molecular mass 13 kDa), which is suitable for studies with CD and NMR spectroscopy. For the wild-type E6AP(C-lobe) authors provided high-quality experimental data including 3D 13C and 15N NOESY spectra (Figure 4), which can be used for evaluation of high-resolution 3D structure of the E6AP(C-lobe).

Author’s response: We thank the reviewer for these kind comments.

However, experimental conditions deserve some criticism, first of all due to the pH of buffer used in experiments close to the isoelectric point (estimated pI 6.94).

Author’s response: We thank the reviewer for this constructive feedback. We agree – the NMR sample could have been collected in an MES buffer at pH 6.0 buffer, similar to our previous publication on ITCH (Beasley et al., 2019) which would have slowed amide exchange to increase solubility and NMR signal to noise. The CD spectra were collected at pH 7.4 and our activity assays were performed at pH 7.5. We would like to point out that the concentration in our E6AP C-lobe wild-type sample, which was collected at pH 7.0, contained 3.5 mM, suggesting that the pH we used was appropriate for our studies. It is also noteworthy that the theoretical wild-type E6AP HECT domain is 5.06, while the E6AP HECT I827K protein is 5.11, which also showed a disruption in ubiquitylation activity. This suggests that I827K substitution is responsible for the observed aggregation and loss of activity and is likely not due to the pH that we used to conduct our experiments.

As demonstrated by authors, the mutation I827K lead to structural changes, which are clearly visible with CD and NMR techniques. Nevertheless, I couldn’t exclude that observed data are artifact due to experimental conditions. The 15N HSQC spectrum for E6AP(C-lobe) I827K mutant (Figure 3B) suggests a strong aggregation phenomenon rather than unfolded protein. To discriminate between these hypotheses, the authors would have to perform additional experiments.

Author’s response: We thank the review for this constructive feedback. Upon further examination of our data, we agree with you that the C-lobe is not completely unfolded and that our NMR spectra and biochemical assays support the loss of activity due to protein aggregation. To address this, we have modified the language in the manuscript to state that the I827K Angelman Syndrome substitution causes the partial unfolding and aggregation of the protein. We also took another look at our previous gel filtration elution profiles during our initial protein purifications and we noted a significant amount of the E6AP C-lobe I827K protein eluted in void volume indicative of protein aggregation.

The short run of structural minimization after I827K substitution shows visible changes in orientation of side-chains of hydrophobic residues, but such dramatic differences were not confirmed experimentally. I could note that I827K mutation increases the pI up to 8.03, which is still very close to pH buffer used in experiments. Further, the K827 together with K829 evidently forms a hydrophilic cluster characterized by positive charge which can destabilize 3D structure of the E6AP(C-lobe). The computer simulations can help understanding that process in details. It’s not surprising that the mutation can facilitate allosteric effects on catalytic C843 resulting in decreased interaction with the polyubiquitination chain.

Author’s response: We thank the reviewer for bringing this to our attention. We agree that the hydrophilic polylysine cluster is very interesting observation and is something that we will be looking into further in a future study.

Summarizing. To discriminate between unfolded or molten globule state, there are some additional experiments required to extract the size of protein in solution, which would help understanding structural changes under I827K mutation. First, we suggest to measure translation diffusion with NMR spectroscopy (DOSY package). To my knowledge, Varian Inova 600 spectrometer is equipped with Performa IV z-gradient unit generated up to 70 G/cm. This is enough to measure diffusion for 13 kDa protein. On the other hand, other experiments can be used, such as the DLS measurements, to estimate size of particles in solution.

Author’s response: We thank the reviewer for this helpful suggestion. As stated above, after reexamining our size exclusion gel filtration data during purification of the E6AP C-lobe I827K protein we noted that a significant amount of the protein eluted in the void volume, indicative of aggregation. The line broadening seen in the E6AP C-lobe I827K sample (Figure 3B) also indicates protein aggregation. We also thank the reviewer for their advice of running translation diffusion experiments, but unfortunately, we are unable to collect these experiments at this time due to research being halted at Clark University and the current shutdown of our NMR facility due to the COVID-19 pandemic.

Minor points. Authors have to be more careful about manuscript preparation. The experimental conditions for the NMR experiments are different in ‘Materials and Methods’ and in Caption to Figure 3. Also the Figure 4A contains a wrong number of W827 (should be W791).

Author’s response: Thank the reviewer for noticing these errors. The buffer was corrected in the caption of Figure 3. We have also fixed the tryptophan label in Figure 4A.

We are very excited about our studies on how E6AP dysfunction contributes to Angelman’s Syndrome and we hope you share our enthusiasm. Thank you again for considering our manuscript for publication in PLOS ONE.

Sincerely,

Donald E. Spratt, Ph.D.

Assistant Professor of Chemistry & Biochemistry

Gustaf H. Carlson School of Chemistry & Biochemistry

Clark University

Decision Letter 1

Michael Massiah

25 Jun 2020

An Angelman Syndrome Substitution in the HECT E3 Ubiquitin Ligase C-terminal Lobe of E6AP Affects Protein Stability and Activity

PONE-D-20-07946R1

Dear Dr. Spratt,

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Kind regards,

Michael Massiah

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

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Reviewer #1: N/A

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Reviewer #1: Yes

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Reviewer #1: Well revised manuscript. The authors answered all questions raised, however, some additional experimental work was not done due to Corona problems. The explanations are given instead.

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Reviewer #1: No

Acceptance letter

Michael Massiah

26 Jun 2020

PONE-D-20-07946R1

An Angelman Syndrome Substitution in the HECT E3 Ubiquitin Ligase C-terminal Lobe of E6AP Affects Protein Stability and Activity

Dear Dr. Spratt:

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    Data Availability Statement

    All relevant data are within the manuscript. All chemical shift assignments and experiments were deposited into the Biological Magnetic Resonance Databank (http://www.bmrb.wisc.edu) under accession code 50084.

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