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Biomolecular NMR Assignments

, Volume 13, Issue 1, pp 15–20 | Cite as

1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH

  • Steven A. Beasley
  • Roela Bardhi
  • Donald E. SprattEmail author
Open Access
Article

Abstract

ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family that regulates numerous cellular functions including inflammatory responses through T-cell activation, cell differentiation, and apoptosis. Known intracellular targets of ITCH-dependent ubiquitylation include receptor proteins, signaling molecules, and transcription factors. The HECT C-terminal lobe of ITCH contains the conserved catalytic cysteine required for the covalent attachment of ubiquitin onto a substrate and polyubiquitin chain assembly. We report here the complete experimentally determined 1H, 13C, and 15N backbone and sidechain resonance assignments for the HECT C-terminal lobe of ITCH (residues 784–903) using heteronuclear, multidimensional NMR spectroscopy. These resonance assignments will be used in future NMR-based studies to examine the role of dynamics and conformational flexibility in HECT-dependent ubiquitylation as well as deciphering the structural and biochemical basis for polyubiquitin chain synthesis and specificity by ITCH.

Keywords

ITCH Atrophin-1-interacting protein 4 Ubiquitin HECT E3 ubiquitin ligase Ubiquitylation NMR spectroscopy 

Abbreviations

HECT

Homologous to E6AP C-terminus

NMR

Nuclear magnetic resonance

Biological context

Ubiquitylation is an important posttranslational modification that maintains cellular health and homeostasis by targeting proteins for proteosomal or autophagic degradation (Cohen-Kaplan et al. 2016). Ubiquitylation occurs through the sequential transfer of ubiquitin between three enzymes—ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3)—ultimately resulting in the covalent attachment of ubiquitin on to a substrate via an isopeptide bond. In contrast to the RING (Really Interesting New Gene) E3 ubiquitin ligases that primarily act as scaffolds by orienting the E2 ~ ubiquitin thiolester complex and target protein for ubiquitin transfer, the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligases play a catalytic role in the final attachment of ubiquitin by forming a thiolester intermediate with ubiquitin before transferring it to a substrate protein (Lorenz 2018; Metzger et al. 2012). The combinatorial effect of ~ 40 human E2 enzymes and hundreds of E3 ligases allows for the observed diversity of ubiquitylation substrate specificity.

In humans, there are 28 members of the HECT E3 ubiquitin ligase family (Scheffner and Kumar 2014) with each containing a conserved ~ 350 residue HECT domain near their C-termini. Each HECT domain is comprised of two lobes—a larger N-terminal lobe responsible for recruiting an E2 ~ ubiquitin complex, and a smaller C-terminal lobe that contains the catalytic cysteine required to covalently attach ubiquitin to its substrates (Lorenz 2018). The HECT E3 ubiquitin ligase family can be further categorized into subfamilies, which includes the most well-examined NEDD4 subfamily. The NEDD4 HECT E3 ubiquitin ligases are comprised of a Ca2+-binding C2 domain, WW domains that bind the PPxY protein–protein interaction motif, as well as the HECT domain required for its ubiquitylation activity. The 3D structures of several NEDD4 HECT domains family members have been solved by X-ray crystallography, either alone or complexed with E2 and/or ubiquitin (Lorenz 2018), however, there are many unanswered questions regarding how these enzymes assemble into multi-protein complexes to synthesize polyubiquitin chains.

ITCH, also known as Atrophin-1-interacting protein 4, is a prominent member of the NEDD4 subfamily that regulates signaling pathways involved in immune-cell differentiation, control of the inflammatory signaling pathways, and apoptosis (Aki et al. 2015). ITCH is an intriguing HECT E3 ubiquitin ligase due to its ability to synthesize different polyubiquitin lysine linkages (i.e. K29, K48, and/or K63-linked) depending on the substrate it modifies, and this ubiquitin linkage specificity is dependent on the last 60 amino acids of the C-terminal lobe of ITCH (Kim and Huibregtse 2009). For example, ITCH has been observed to build K29-linked polyubiquitin chains on the transmembrane receptor protein Notch that leads to Notch undergoing endocytosis and lysosomal degradation (Chastagner et al. 2008). ITCH knockout mice display an ‘Itchy’ phenotype caused by inflammatory dysregulation due to unfettered Notch signaling (Chastagner et al. 2008; Matesic et al. 2008). ITCH negatively regulates the Hippo tumor suppressor pathway by building a K48-linked polyubiquitin chain on of the serine/threonine kinase LATS1, a tumor growth inhibitor that induces G2-M arrest and apoptosis (Salah et al. 2014). ITCH also controls intracellular concentrations of p63 and p73, members of the tumor suppressor protein family, by K48-polyubiquitylation to target these oncoproteins for proteosomal degradation (Melino et al. 2008). K63-linked ITCH-dependent polyubiquitination has also been observed for the transcription factor p45/NF-E2 causing it to migrate out of the nucleus into the cytoplasm and thus inactivating p45/NF-E2 (Lee et al. 2008).

Presently there is no structural rationale for how ITCH is capable of building these different polyubiquitin chain types and the catalytic mechanism for ITCH is currently unknown. Here we report the complete backbone and sidechain 1H, 13C, and 15N resonance assignments for the catalytic C-terminal lobe of ITCH (residues 784–903) using 3D heteronuclear NMR spectroscopy. These resonance assignments will enable future structural and mechanistic studies to better understand ITCH-dependent ubiquitylation.

Methods and experiments

Protein expression and purification

The human HECT C-terminal lobe of ITCH (Uniprot: Q96J02, residues 784–903) with C835S and C855S substitutions was synthesized and codon-optimized by DNA2.0 (Newark, CA, USA) and cloned into an ampicillin resistant T7-inducible vector with an N-terminal polyhistidine tag (His6-tag) followed by a TEV protease cleavage site (ENLYFQ). The His6-TEV-ITCH C-terminal lobe construct was transformed into E. coli BL21(DE3) RIL+ and grown at 37 °C in M9 media (2 × 1 L) supplemented with 15NH4Cl (1 g/L), 13C6-glucose (2 g/L), 100 mg/L ampicillin and 34 mg/L chloramphenicol. When the culture reached an OD600 of 0.8, the cultures were induced with 1 mM IPTG at 16 °C for 20 h. The cells were harvested by centrifugation at 6000×g for 10 min using a Sorvall LYNX 4000 superspeed centrifuge with a Fiberlite F 10 − 4 × 1000 LEX Carbon Fiber rotor (Thermo-Fisher). Cell pellets were resuspended in 20 mL of wash buffer (50 mM Na2HPO4 pH 8.0, 300 mM NaCl, 10 mM imidazole) with 1 mM PMSF and an EDTA-free protease inhibitor mini tablet (Pierce), lysed using an Emulsiflex-C5 homogenizer (Avestin, Ottawa, ON, Canada), and clarified by ultracentrifugation using a Optima L-80 XP ultracentrifuge with a 70.1 Ti rotor (Beckman-Coulter) at 41,000 rpm for 40 min. The clarified supernatant was then applied to 5 mL of HisPur Ni–NTA resin (Thermo-Fisher) pre-equilibrated with wash buffer at a flow rate of 0.5 mL/min. After the resin was washed with 25 column volumes of wash buffer, the protein was eluted with 20 mL of elution buffer (50 mM Na2HPO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). Fractions containing eluted protein were pooled and incubated with recombinant TEV protease for 1 h at 25 °C (1 mg TEV/50 mg eluted protein) to cleave the His6-tag, then dialyzed against wash buffer stirring overnight at 4 °C. The TEV cleaved protein was then reapplied to 5 mL of HisPur Ni–NTA resin at a flow rate of 0.5 mL/min and the flowthrough containing 13C–15N-labeled ITCH C-terminal lobe was collected and pooled. The protein was then concentrated using a Amicon 15 mL centrifugal filter with a 10 kDa MWCO (Millipore) and loaded onto a HiLoad 16/60 Superdex75 column equilibrated with 20 mM MES, 120 mM NaCl, 1 mM EDTA, 2 mM TCEP, pH 6.0 at a flow rate of 1 mL/min using an ÄKTA pure 25L FPLC (GE Healthcare Life Sciences). Fractions containing the purified 13C–15N-labeled ITCH C-terminal lobe, as assessed by SDS-PAGE, were pooled and concentrated. The resulting ITCH C-terminal lobe protein had an additional “GS” at its N-terminus as a result of its cloning and TEV cleavage. After purification, the concentration of the protein was determined using the Bradford assay (Bio-Rad) or a A280 using a Nanodrop One C UV/Vis spectrophotometer (Thermo-Fisher).

NMR spectroscopy

The NMR samples used for resonance assignment of 13C–15N-labeled human ITCH C-terminal lobe were prepared in 20 mM MES, 120 mM NaCl, 1 mM EDTA, 2 mM TCEP, 10% D2O/90% H2O at pH 6.0. The samples were concentrated to a final volume of 600 μL and transferred to a 5 mm O.D. thin walled NMR tube (New-Era). Imidazole (1.6 mM) was added as an internal pH indicator to monitor the pH of the sample during data acquisition (Baryshnikova et al. 2008).

All NMR data were collected at 25 °C using 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 Sackler Sciences Center at Clark University. Backbone assignments and aliphatic side chain assignments were determined using the following standard pulse sequences in the Varian Biopack in VnmrJ 3.0: 1H–15N HSQC, aliphatic 1H–13C HSQC, HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH, aliphatic HCCH-TOCSY, C(CO)NH, H(CCO)NH, and 1H–15N NOESY. Aromatic sidechain assignments were determined using an aromatic 13C-HSQC, HBCBCGCDHD and HBCBCGCDCEHE experiments in combination with an aromatic HCCH-TOCSY and aromatic 13C-NOESY experiments. All data were processed using NMRPipe and NMRDraw (Delaglio et al. 1995) and analyzed using NMRViewJ (Johnson and Blevins 1994). All of the relevant peak lists and the complete 1H, 13C, and 15N backbone and sidechain chemical shift assignments have been deposited into the Biological Magnetic Resonance Databank (http://www.bmrb.wisc.edu) under ascension code 27477.

Assignments and data deposition

The non-artifact and non-proline amide protons (113/115; 98.26%), backbone atoms (350/360; 97.22%) and 1H chemical shift assignments of the Hα (119/120, 99.17%) and Hβ (201/203, 99.01%) were definitively assigned. Every Cα and Cβ resonance were assigned except for the N-terminal artifact glycine and P878 (120/122, 98.36%). Aliphatic sidechain resonances of Cγ (60/62, 96.77%), Cδ (29/30, 96.67%), and Cε (13/13, 100%) were also assigned. Almost all nitrogen atoms—excluding the lysine, arginine, and histidine sidechain and proline backbone nitrogen atoms—were definitively assigned (130/132; 98.48%). The missing amide peaks for K861 and W864 could not be assigned possibly due to fast amide exchange with the solvent and/or high flexibility of the region. There were no unassigned peaks visible on the well-dispersed 1H–15N HSQC spectrum (Fig. 1). One residue of particular interest that will be followed in future chemical shift perturbation experiments is the catalytic cysteine of ITCH (C871) as it has distinct Cβ peaks on the 1H–13C HSQC whose chemical shift indicates that it is reduced (Kornhaber et al. 2006) as would be required for its ubiquitylation activity.

Fig. 1

Assigned observable 1H–15N HSQC spectrum of the human HECT C-terminal lobe of ITCH (residues 784–903, 2.6 mM). The spectrum is labeled according to the one-amino acid code and residue number of the human ITCH sequence. The NMR sample contained 13C and 15N-isotopically enriched ITCH in 20 mM MES pH 6.0, 120 mM NaCl, 1 mM EDTA, and 10% D2O. Data was collected on a Varian Inova 600-MHz NMR spectrometer at 25 °C. Peaks corresponding to asparagine and glutamine side chain amides are connected with a horizontal line

ITCH is a member of the NEDD4 subfamily of HECT E3 ubiquitin ligases that shows a high degree of sequence conservation (Fig. 2a) with many of these conserved residues found within the hydrophobic core of the protein. For example, there are exceptional upfield peaks of the W813 aromatic side chain in both the 1H–15N HSQC and aromatic 1H–13C HSQC, likely due to shielding effect within the hydrophobic core surrounded by three proximal aromatic residues (i.e. W793, Y799, and F812; Fig. 2b). Intriguingly, there is one exceptional tryptophan (W864) on the surface that is found only in ITCH, as well as WWP1 (W883) and WWP2 (W823). This residue, which resides on the same face of the protein as the catalytic cysteine, deserves further attention as the mechanism of ubiquitin chain building and lysine-linkage specificity depends on these subtle differences between the different NEDD4 subfamily members. The secondary structure based on the chemical shift index (CSI) 3.0 web server analysis of chemical shifts (Hafsa et al. 2015) is in good agreement with the known PDB structures of ITCH (Zhang et al. 2016) and other HECT domain C-terminal lobes with an αβα2β3α organization indicating that the protein is well folded and in the correct structural conformation (Fig. 3).

Fig. 2

a Multiple sequence alignment of the C-terminal lobes of the NEDD4 HECT E3 ubiquitin ligase subfamily. The sequence alignment was performed using T-Coffee (Notredame et al. 2000) followed by manual curation in Jalview (Waterhouse et al. 2009). The α-helices and β-sheets based on the crystal structure of ITCH are shown as cylinders and arrows, respectively. Absolutely conserved residues are marked with an asterisk. b Structure of ITCH C-terminal lobe (PDB:3TUG) highlighting the catalytic cysteine C871 (red), as well as the W813 (cyan), surrounded by aromatic residues W793, Y799, and F812 (yellow). The solvent exposed tryptophan W864 is shown in magenta. Noteworthy residues discussed in the text are also highlighted in panel A using the same color scheme

Fig. 3

Predicted secondary structural regions of the ITCH C-terminal lobe. The probability plot was made by inputting the experimentally determined resonance assignments for ITCH into the online webserver CSI 3.0 (Hafsa et al. 2015). The propensity to form an a-helix or b-sheet are denoted in red and blue, respectively

In conclusion, we present the complete backbone and side chain resonance assignments of the catalytic HECT C-terminal lobe of ITCH. These resonance assignments will be used to examine the role of inherent conformational flexibility within the C-terminal lobe of ITCH that will help us decipher how ITCH is capable of building K29, K48, and K63-linked polyubiquitin chains on its diverse intracellular substrates.

Notes

Acknowledgements

The authors would like to thank Dr. Guoxing Lin for his assistance in setting up experiments and for maintaining the 600 MHz NMR spectrometer housed in the Carlson School of Chemistry and Biochemistry at Clark University. This work was supported by a grant from the National Institutes of Health (R15GM126432) and start-up funds from Clark University.

References

  1. Aki D, Zhang W, Liu YC (2015) The E3 ligase Itch in immune regulation and beyond. Immunol Rev 266:6–26.  https://doi.org/10.1111/imr.12301 CrossRefGoogle Scholar
  2. Baryshnikova OK, Williams TC, Sykes BD (2008) Internal pH indicators for biomolecular. NMR J Biomol NMR 41:5–7.  https://doi.org/10.1007/s10858-008-9234-6 CrossRefGoogle Scholar
  3. Chastagner P, Israel A, Brou C (2008) AIP4/Itch regulates Notch receptor degradation in the absence of ligand. PLoS ONE 3:e2735.  https://doi.org/10.1371/journal.pone.0002735 ADSCrossRefGoogle Scholar
  4. Cohen-Kaplan V, Livneh I, Avni N, Cohen-Rosenzweig C, Ciechanover A (2016) The ubiquitin-proteasome system and autophagy: coordinated and independent activities. Int J Biochem Cell Biol 79:403–418.  https://doi.org/10.1016/j.biocel.2016.07.019 CrossRefGoogle Scholar
  5. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  6. Hafsa NE, Arndt D, Wishart DS (2015) CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts. Nucleic Acids Res 43:W370–W377.  https://doi.org/10.1093/nar/gkv494 CrossRefGoogle Scholar
  7. Johnson BA, Blevins RA (1994) NMR view: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614.  https://doi.org/10.1007/BF00404272 CrossRefGoogle Scholar
  8. Kim HC, Huibregtse JM (2009) Polyubiquitination by HECT E3s and the determinants of chain type specificity. Mol Cell Biol 29:3307–3318.  https://doi.org/10.1128/MCB.00240-09 CrossRefGoogle Scholar
  9. Kornhaber GJ, Snyder D, Moseley HN, Montelione GT (2006) Identification of zinc-ligated cysteine residues based on 13Calpha and 13Cbeta chemical shift data. J Biomol NMR 34:259–269.  https://doi.org/10.1007/s10858-006-0027-5 CrossRefGoogle Scholar
  10. Lee TL, Shyu YC, Hsu TY, Shen CK (2008) Itch regulates p45/NF-E2 in vivo by Lys63-linked ubiquitination. Biochem Biophys Res Commun 375:326–330.  https://doi.org/10.1016/j.bbrc.2008.07.164 CrossRefGoogle Scholar
  11. Lorenz S (2018) Structural mechanisms of HECT-type ubiquitin ligases. Biol Chem 399:127–145.  https://doi.org/10.1515/hsz-2017-0184 CrossRefGoogle Scholar
  12. Matesic LE, Copeland NG, Jenkins NA (2008) Itchy mice: the identification of a new pathway for the development of autoimmunity. Curr Top Microbiol Immunol 321:185–200Google Scholar
  13. Melino G et al (2008) Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death Differ 15:1103–1112.  https://doi.org/10.1038/cdd.2008.60 CrossRefGoogle Scholar
  14. Metzger MB, Hristova VA, Weissman AM (2012) HECT and RING finger families of E3 ubiquitin ligases at a glance. J Cell Sci 125:531–537.  https://doi.org/10.1242/jcs.091777 CrossRefGoogle Scholar
  15. Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217.  https://doi.org/10.1006/jmbi.2000.4042 CrossRefGoogle Scholar
  16. Salah Z, Itzhaki E, Aqeilan RI (2014) The ubiquitin E3 ligase ITCH enhances breast tumor progression by inhibiting the Hippo tumor suppressor pathway. Oncotarget 5:10886–10900.  https://doi.org/10.18632/oncotarget.2540 CrossRefGoogle Scholar
  17. Scheffner M, Kumar S (2014) Mammalian HECT ubiquitin-protein ligases: biological and pathophysiological aspects. Biochim Biophys Acta 1843:61–74.  https://doi.org/10.1016/j.bbamcr.2013.03.024 CrossRefGoogle Scholar
  18. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191.  https://doi.org/10.1093/bioinformatics/btp033 CrossRefGoogle Scholar
  19. Zhang W et al (2016) System-wide modulation of HECT E3 ligases with selective ubiquitin variant probes. Mol Cell 62:121–136.  https://doi.org/10.1016/j.molcel.2016.02.005 CrossRefGoogle Scholar

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Gustaf H. Carlson School of Chemistry and BiochemistryClark UniversityWorcesterUSA

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