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

1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin... ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Ter- minus) 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 1 13 15 report here the complete experimentally determined H, C, and N 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 flex- ibility 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 Interesting New Gene) E3 ubiquitin ligases that primarily HECT Homologous to E6AP C-terminus act as scaffolds by orienting the E2 ~ ubiquitin thiolester NMR Nuclear magnetic resonance 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 Biological context forming a thiolester intermediate with ubiquitin before trans- ferring it to a substrate protein (Lorenz 2018; Metzger et al. Ubiquitylation is an important posttranslational modifica- 2012). The combinatorial effect of ~ 40 human E2 enzymes tion that maintains cellular health and homeostasis by tar- and hundreds of E3 ligases allows for the observed diversity geting proteins for proteosomal or autophagic degradation of ubiquitylation substrate specificity. (Cohen-Kaplan et al. 2016). Ubiquitylation occurs through In humans, there are 28 members of the HECT E3 ubiq- the sequential transfer of ubiquitin between three enzymes— uitin ligase family (Scheffner and Kumar 2014) with each ubiquitin-activating enzyme (E1), ubiquitin-conjugating containing a conserved ~ 350 residue HECT domain near enzyme (E2), and ubiquitin ligase (E3)—ultimately result- their C-termini. Each HECT domain is comprised of two ing in the covalent attachment of ubiquitin on to a substrate lobes—a larger N-terminal lobe responsible for recruit- via an isopeptide bond. In contrast to the RING (Really ing an E2 ~ ubiquitin complex, and a smaller C-terminal lobe that contains the catalytic cysteine required to cova- lently attach ubiquitin to its substrates (Lorenz 2018). The Steven A. Beasley and Roela Bardhi have contributed equally to HECT E3 ubiquitin ligase family can be further categorized this manuscript. into subfamilies, which includes the most well-examined NEDD4 subfamily. The NEDD4 HECT E3 ubiquitin ligases * Donald E. Spratt 2+ are comprised of a Ca -binding C2 domain, WW domains dspratt@clarku.edu that bind the PPxY protein–protein interaction motif, as well Gustaf H. Carlson School of Chemistry and Biochemistry, as the HECT domain required for its ubiquitylation activity. Clark University, 950 Main St., Worcester, MA 01610, USA Vol.:(0123456789) 1 3 16 S. A. Beasley et al. The 3D structures of several NEDD4 HECT domains family (Newark, CA, USA) and cloned into an ampicillin resist- members have been solved by X-ray crystallography, either ant T7-inducible vector with an N-terminal polyhistidine alone or complexed with E2 and/or ubiquitin (Lorenz 2018), tag (His -tag) followed by a TEV protease cleavage site however, there are many unanswered questions regarding (ENLYFQ). The His -TEV-ITCH C-terminal lobe con- how these enzymes assemble into multi-protein complexes struct was transformed into E. coli BL21(DE3) RIL+ and to synthesize polyubiquitin chains. grown at 37 °C in M9 media (2 × 1 L) supplemented with 15 13 ITCH, also known as Atrophin-1-interacting protein 4, NH Cl (1 g/L), C -glucose (2 g/L), 100 mg/L ampicillin 4 6 is a prominent member of the NEDD4 subfamily that regu- and 34 mg/L chloramphenicol. When the culture reached an lates signaling pathways involved in immune-cell differen- OD600 of 0.8, the cultures were induced with 1 mM IPTG at tiation, control of the inflammatory signaling pathways, and 16 °C for 20 h. The cells were harvested by centrifugation at apoptosis (Aki et al. 2015). ITCH is an intriguing HECT E3 6000×g for 10 min using a Sorvall LYNX 4000 superspeed ubiquitin ligase due to its ability to synthesize different poly - centrifuge with a Fiberlite F 10 − 4 × 1000 LEX Carbon ubiquitin lysine linkages (i.e. K29, K48, and/or K63-linked) Fiber rotor (Thermo-Fisher). Cell pellets were resuspended depending on the substrate it modifies, and this ubiquitin in 20 mL of wash buffer (50 mM Na HPO pH 8.0, 300 mM 2 4 linkage specificity is dependent on the last 60 amino acids NaCl, 10 mM imidazole) with 1 mM PMSF and an EDTA- of the C-terminal lobe of ITCH (Kim and Huibregtse 2009). free protease inhibitor mini tablet (Pierce), lysed using an For example, ITCH has been observed to build K29-linked Emulsiflex-C5 homogenizer (Avestin, Ottawa, ON, Canada), polyubiquitin chains on the transmembrane receptor protein and clarified by ultracentrifugation using a Optima L-80 Notch that leads to Notch undergoing endocytosis and lyso- XP ultracentrifuge with a 70.1 Ti rotor (Beckman-Coulter) somal degradation (Chastagner et al. 2008). ITCH knockout at 41,000 rpm for 40 min. The clarified supernatant was mice display an ‘Itchy’ phenotype caused by inflammatory then applied to 5 mL of HisPur Ni–NTA resin (Thermo- dysregulation due to unfettered Notch signaling (Chast- Fisher) pre-equilibrated with wash buffer at a flow rate of agner et al. 2008; Matesic et al. 2008). ITCH negatively 0.5 mL/min. After the resin was washed with 25 column regulates the Hippo tumor suppressor pathway by building volumes of wash buffer, the protein was eluted with 20 mL a K48-linked polyubiquitin chain on of the serine/threonine of elution buffer (50 mM Na HPO pH 8.0, 300 mM NaCl, 2 4 kinase LATS1, a tumor growth inhibitor that induces G2-M 250 mM imidazole). Fractions containing eluted protein arrest and apoptosis (Salah et al. 2014). ITCH also controls were pooled and incubated with recombinant TEV protease intracellular concentrations of p63 and p73, members of the for 1 h at 25 °C (1 mg TEV/50 mg eluted protein) to cleave tumor suppressor protein family, by K48-polyubiquitylation the His -tag, then dialyzed against wash buffer stirring over - to target these oncoproteins for proteosomal degradation night at 4 °C. The TEV cleaved protein was then reapplied (Melino et  al. 2008). K63-linked ITCH-dependent poly- to 5 mL of HisPur Ni–NTA resin at a flow rate of 0.5 mL/ 13 15 ubiquitination has also been observed for the transcription min and the flowthrough containing C– N-labeled ITCH factor p45/NF-E2 causing it to migrate out of the nucleus C-terminal lobe was collected and pooled. The protein was into the cytoplasm and thus inactivating p45/NF-E2 (Lee then concentrated using a Amicon 15 mL centrifugal filter et al. 2008). with a 10 kDa MWCO (Millipore) and loaded onto a HiLoad Presently there is no structural rationale for how ITCH 16/60 Superdex75 column equilibrated with 20 mM MES, is capable of building these different polyubiquitin chain 120  mM NaCl, 1  mM EDTA, 2  mM TCEP, pH 6.0  at a types and the catalytic mechanism for ITCH is currently flow rate of 1 mL/min using an ÄKTA pure 25L FPLC (GE unknown. Here we report the complete backbone and side- Healthcare Life Sciences). Fractions containing the puri- 1 13 15 13 15 chain H, C, and N resonance assignments for the cata- fied C– N-labeled ITCH C-terminal lobe, as assessed by lytic C-terminal lobe of ITCH (residues 784–903) using 3D SDS-PAGE, were pooled and concentrated. The resulting heteronuclear NMR spectroscopy. These resonance assign- ITCH C-terminal lobe protein had an additional “GS” at its ments will enable future structural and mechanistic studies N-terminus as a result of its cloning and TEV cleavage. After to better understand ITCH-dependent ubiquitylation. purification, the concentration of the protein was determined using the Bradford assay (Bio-Rad) or a A using a Nan- odrop One C UV/Vis spectrophotometer (Thermo-Fisher). Methods and experiments NMR spectroscopy Protein expression and purification The NMR samples used for resonance assignment of 13 15 The human HECT C-terminal lobe of ITCH (Uniprot: C– N-labeled human ITCH C-terminal lobe were pre- Q96J02, residues 784–903) with C835S and C855S substi- pared in 20 mM MES, 120 mM NaCl, 1 mM EDTA, 2 mM tutions was synthesized and codon-optimized by DNA2.0 TCEP, 10% DO/90% H O at pH 6.0. The samples were 2 2 1 3 1 13 15 H, C, and  N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin… 17 concentrated to a final volume of 600 μL and transferred to due to fast amide exchange with the solvent and/or high flex- a 5 mm O.D. thin walled NMR tube (New-Era). Imidazole ibility of the region. There were no unassigned peaks vis- 1 15 (1.6 mM) was added as an internal pH indicator to monitor ible on the well-dispersed H– N HSQC spectrum (Fig. 1). the pH of the sample during data acquisition (Baryshnikova One residue of particular interest that will be followed in et al. 2008). future chemical shift perturbation experiments is the cata- All NMR data were collected at 25 °C using a Varian lytic cysteine of ITCH (C871) as it has distinct Cβ peaks on 1 13 Inova 600 MHz 4-channel solution-state NMR Spectrometer the H– C HSQC whose chemical shift indicates that it is equipped with a 5-mm PFG triple-resonance probe housed reduced (Kornhaber et al. 2006) as would be required for its and maintained in the Sackler Sciences Center at Clark ubiquitylation activity. University. Backbone assignments and aliphatic side chain ITCH is a member of the NEDD4 subfamily of HECT assignments were determined using the following standard E3 ubiquitin ligases that shows a high degree of sequence 1 15 pulse sequences in the Varian Biopack in VnmrJ 3.0: H– N conservation (Fig. 2a) with many of these conserved resi- 1 13 HSQC, aliphatic H– C HSQC, HNCO, HN(CA)CO, dues found within the hydrophobic core of the protein. For HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH, ali- example, there are exceptional upfield peaks of the W813 1 15 1 15 phatic HCCH-TOCSY, C(CO)NH, H(CCO)NH, and H– N aromatic side chain in both the H– N HSQC and aromatic 1 13 NOESY. Aromatic sidechain assignments were determined H– C HSQC, likely due to shielding effect within the using an aromatic C-HSQC, HBCBCGCDHD and HBCB- hydrophobic core surrounded by three proximal aromatic CGCDCEHE experiments in combination with an aromatic residues (i.e. W793, Y799, and F812; Fig. 2b). Intriguingly, HCCH-TOCSY and aromatic C-NOESY experiments. All there is one exceptional tryptophan (W864) on the surface data were processed using NMRPipe and NMRDraw (Dela- that is found only in ITCH, as well as WWP1 (W883) and glio et al. 1995) and analyzed using NMRViewJ (Johnson WWP2 (W823). This residue, which resides on the same and Blevins 1994). All of the relevant peak lists and the face of the protein as the catalytic cysteine, deserves further 1 13 15 complete H, C, and N backbone and sidechain chemical attention as the mechanism of ubiquitin chain building and shift assignments have been deposited into the Biological lysine-linkage specificity depends on these subtle differences Magnetic Resonance Databank (http://www.bmrb.wisc.edu) between the different NEDD4 subfamily members. The sec- under ascension code 27477. ondary structure based on the chemical shift index (CSI) 3.0 web server analysis of chemical shifts (Hafsa et al. 2015) is Assignments and data deposition in good agreement with the known PDB structures of ITCH (Zhang et al. 2016) and other HECT domain C-terminal The non-artifact and non-proline amide protons (113/115; lobes with an αβα β α organization indicating that the pro- 2 3 98.26%), backbone atoms (350/360; 97.22%) and H chemi- tein is well folded and in the correct structural conformation cal shift assignments of the Hα (119/120, 99.17%) and Hβ (Fig. 3). (201/203, 99.01%) were definitively assigned. Every Cα In conclusion, we present the complete backbone and side and Cβ resonance were assigned except for the N-terminal chain resonance assignments of the catalytic HECT C-ter- artifact glycine and P878 (120/122, 98.36%). Aliphatic minal lobe of ITCH. These resonance assignments will be sidechain resonances of Cγ (60/62, 96.77%), Cδ (29/30, used to examine the role of inherent conformational flex- 96.67%), and Cε (13/13, 100%) were also assigned. Almost ibility within the C-terminal lobe of ITCH that will help us all nitrogen atoms—excluding the lysine, arginine, and his- decipher how ITCH is capable of building K29, K48, and tidine sidechain and proline backbone nitrogen atoms—were K63-linked polyubiquitin chains on its diverse intracellular definitively assigned (130/132; 98.48%). The missing amide substrates. peaks for K861 and W864 could not be assigned possibly 1 3 18 S. A. Beasley et al. 100.0 105.0 110.0 115.0 120.0 125.0 130.0 10.0 9.0 8.0 7.0 6.0 H Chemical Shift (ppm) 1 15 Fig. 1 Assigned observable H– N HSQC spectrum of the human 6.0, 120 mM NaCl, 1 mM EDTA, and 10% D O. Data was collected HECT C-terminal lobe of ITCH (residues 784–903, 2.6  mM). The on a Varian Inova 600-MHz NMR spectrometer at 25 °C. Peaks cor- spectrum is labeled according to the one-amino acid code and resi- responding to asparagine and glutamine side chain amides are con- due number of the human ITCH sequence. The NMR sample con- nected with a horizontal line 13 15 tained C and N-isotopically enriched ITCH in 20  mM MES pH 1 3 N Chemical Shift (ppm) 1 13 15 H, C, and  N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin… 19 AB * * ** *** * * * ******** ** ITCH/784-849 GMQEIDLNDWQRHAIYRH-YARTSKQIMWFWQFVKEIDNEKRMRLLQFVTGTCRLPVGGFADLMGSN F812 NEDD4/1198-1264 GLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAELYGSN NED4L/854-920 GLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSN HECW1/1486-1552 GTAEIDLNDWRNNTEYRGGYHDGHLVIRWFWAAVERFNNEQRLRLLQFVTGTSSVPYEGFAALRGSN HECW2/1452-1518 GTAEIDLSDWRNNTEYRGGYHDNHIVIRWFWAAVERFNNEQRLRLLQFVTGTSSIPYEGFASLRGSN W864 WWP1/803-868 GMQEVDLADWQRNTVYRH-YTRNSKQIIWFWQFVKETDNEVRMRLLQFVTGTCRLPLGGFAELMGSN WWP2/751-808 GMQEIDMSDWQKSTIYRH-YTKNSKQIQWFWQVVKEMDNEKRIRLLQFVTGTCRLPVGGFAELIGSN C-terminus SMURF1/634-699 GLDKIDLNDWKSNTRLKHCVAD-SNIVRWFWQAVETFDEERRARLLQFVTGSTRVPLQGFKALQGST W813 SMURF2/628-689 GLGKIDVNDWKVNTRLKHCTPD-SNIVKWFWKAVEFFDEERRARLLQFVTGSSRVPLQGFKALQ--- Y799 * ** ****** * *** * ** * * * * ITCH/850-903 ---GPQKFCIEKVGK-ENWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGF-GQE N-terminus NEDD4/1265-1319 ---GPQSFTVEQWGT-PEKLPRAHTCFNRLDLPPYESFEELWDKLQMAIENTQGFDGVD NED4L/921-975 ---GPQLFTIEQWGS-PEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVD HECW1/1553-1606 ---GLRRFCIEKWGK-ITSLPRAHTCFNRLDLPPYPSYSMLYEKLLTAVEETSTF-GLE W793 C871 HECW2/1519-1572 ---GPRRFCVEKWGK-ITALPRAHTCFNRLDLPPYPSFSMLYEKLLTAVEETSTF-GLE WWP1/869-922 ---GPQKFCIEKVGK-DTWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGF-GQE WWP2/809-870 ---GPQKFCIDKVGK-ETWLPRSHTCFNRLDLPPYKSYEQLREKLLYAIEETEGF-GQE SMURF1/700-757 GAAGPRLFTIHLIDANTDNLPKAHTCFNRIDIPPYESYEKLYEKLLTAVEETCGF-AVE SMURF2/690-748 GAAGPRLFTIHQIDACTNNLPKAHTCFNRIDIPPYESYEKLYEKLLTAIEETCGF-AVE Fig. 2 a Multiple sequence alignment of the C-terminal lobes of the are marked with an asterisk. b Structure of ITCH C-terminal lobe NEDD4 HECT E3 ubiquitin ligase subfamily. The sequence align- (PDB:3TUG) highlighting the catalytic cysteine C871 (red), as well ment was performed using T-Coffee (Notredame et al. 2000) followed as the W813 (cyan), surrounded by aromatic residues W793, Y799, by manual curation in Jalview (Waterhouse et al. 2009). The α-helices and F812 (yellow). The solvent exposed tryptophan W864 is shown and β-sheets based on the crystal structure of ITCH are shown as in magenta. Noteworthy residues discussed in the text are also high- cylinders and arrows, respectively. Absolutely conserved residues lighted in panel A using the same color scheme Fig. 3 Predicted secondary 1.0 structural regions of the ITCH C-terminal lobe. The probabil- ity plot was made by inputting the experimentally determined 0.8 resonance assignments for ITCH into the online webserver CSI 3.0 (Hafsa et al. 2015). The 0.6 propensity to form an a-helix or b-sheet are denoted in red and blue, respectively 0.4 0.2 791 801 811 821 831 841 851 861 871 881 891901 Residue Acknowledgements The authors would like to thank Dr. Guoxing Lin References for his assistance in setting up experiments and for maintaining the 600 MHz NMR spectrometer housed in the Carlson School of Chem- Aki D, Zhang W, Liu YC (2015) The E3 ligase Itch in immune regula- istry and Biochemistry at Clark University. This work was supported tion and beyond. Immunol Rev 266:6–26. https://doi.or g/10.1111/ by a grant from the National Institutes of Health (R15GM126432) and imr.12301 start-up funds from Clark University. Baryshnikova OK, Williams TC, Sykes BD (2008) Internal pH indica- tors for biomolecular. NMR J Biomol NMR 41:5–7. https ://doi. Open Access This article is distributed under the terms of the Crea- org/10.1007/s1085 8-008-9234-6 tive Commons Attribution 4.0 International License (http://creat iveco Chastagner P, Israel A, Brou C (2008) AIP4/Itch regulates Notch recep- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tor degradation in the absence of ligand. PLoS ONE 3:e2735. tion, and reproduction in any medium, provided you give appropriate https ://doi.org/10.1371/journ al.pone.00027 35 credit to the original author(s) and the source, provide a link to the Cohen-Kaplan V, Livneh I, Avni N, Cohen-Rosenzweig C, Ciechano- Creative Commons license, and indicate if changes were made. ver A (2016) The ubiquitin-proteasome system and autophagy: 1 3 Probability 20 S. A. Beasley et al. coordinated and independent activities. Int J Biochem Cell Biol Matesic LE, Copeland NG, Jenkins NA (2008) Itchy mice: the identi- 79:403–418. https ://doi.org/10.1016/j.bioce l.2016.07.019 fication of a new pathway for the development of autoimmunity. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) Curr Top Microbiol Immunol 321:185–200 NMRPipe: a multidimensional spectral processing system based Melino G et al (2008) Itch: a HECT-type E3 ligase regulating immu- on UNIX pipes. J Biomol NMR 6:277–293 nity, skin and cancer. Cell Death Differ 15:1103–1112. https://doi. Hafsa NE, Arndt D, Wishart DS (2015) CSI 3.0: a web server for org/10.1038/cdd.2008.60 identifying secondary and super-secondary structure in proteins Metzger MB, Hristova VA, Weissman AM (2012) HECT and RING using NMR chemical shifts. Nucleic Acids Res 43:W370–W377. finger families of E3 ubiquitin ligases at a glance. J Cell Sci https ://doi.org/10.1093/nar/gkv49 4 125:531–537. https ://doi.org/10.1242/jcs.09177 7 Johnson BA, Blevins RA (1994) NMR view: a computer program Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for the visualization and analysis of NMR data. J Biomol NMR for fast and accurate multiple sequence alignment. J Mol Biol 4:603–614. https ://doi.org/10.1007/BF004 04272 302:205–217. https ://doi.org/10.1006/jmbi.2000.4042 Kim HC, Huibregtse JM (2009) Polyubiquitination by HECT E3s Salah Z, Itzhaki E, Aqeilan RI (2014) The ubiquitin E3 ligase ITCH and the determinants of chain type specificity. Mol Cell Biol enhances breast tumor progression by inhibiting the Hippo tumor 29:3307–3318. https ://doi.org/10.1128/MCB.00240 -09 suppressor pathway. Oncotarget 5:10886–10900. https ://doi. Kornhaber GJ, Snyder D, Moseley HN, Montelione GT (2006) Iden-org/10.18632 /oncot arget .2540 tification of zinc-ligated cysteine residues based on 13Calpha Scheffner M, Kumar S (2014) Mammalian HECT ubiquitin-protein and 13Cbeta chemical shift data. J Biomol NMR 34:259–269. ligases: biological and pathophysiological aspects. Biochim Biophys https ://doi.org/10.1007/s1085 8-006-0027-5 Acta 1843:61–74. https ://doi.org/10.1016/j.bbamc r.2013.03.024 Lee TL, Shyu YC, Hsu TY, Shen CK (2008) Itch regulates p45/ Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) NF-E2 in vivo by Lys63-linked ubiquitination. Biochem Bio- Jalview version 2—a multiple sequence alignment editor and phys Res Commun 375:326–330. https ://doi.or g/10.1016/j. analysis workbench. Bioinformatics 25:1189–1191. https ://doi. bbrc.2008.07.164org/10.1093/bioin forma tics/btp03 3 Lorenz S (2018) Structural mechanisms of HECT-type ubiquitin Zhang W et al (2016) System-wide modulation of HECT E3 ligases ligases. Biol Chem 399:127–145. https ://doi.or g/10.1515/ with selective ubiquitin variant probes. Mol Cell 62:121–136. hsz-2017-0184https ://doi.org/10.1016/j.molce l.2016.02.005 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

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

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Springer Journals
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Copyright © 2018 by The Author(s)
Subject
Physics; Biological and Medical Physics, Biophysics; Polymer Sciences; Biochemistry, general
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10.1007/s12104-018-9843-2
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Abstract

ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Ter- minus) 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 1 13 15 report here the complete experimentally determined H, C, and N 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 flex- ibility 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 Interesting New Gene) E3 ubiquitin ligases that primarily HECT Homologous to E6AP C-terminus act as scaffolds by orienting the E2 ~ ubiquitin thiolester NMR Nuclear magnetic resonance 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 Biological context forming a thiolester intermediate with ubiquitin before trans- ferring it to a substrate protein (Lorenz 2018; Metzger et al. Ubiquitylation is an important posttranslational modifica- 2012). The combinatorial effect of ~ 40 human E2 enzymes tion that maintains cellular health and homeostasis by tar- and hundreds of E3 ligases allows for the observed diversity geting proteins for proteosomal or autophagic degradation of ubiquitylation substrate specificity. (Cohen-Kaplan et al. 2016). Ubiquitylation occurs through In humans, there are 28 members of the HECT E3 ubiq- the sequential transfer of ubiquitin between three enzymes— uitin ligase family (Scheffner and Kumar 2014) with each ubiquitin-activating enzyme (E1), ubiquitin-conjugating containing a conserved ~ 350 residue HECT domain near enzyme (E2), and ubiquitin ligase (E3)—ultimately result- their C-termini. Each HECT domain is comprised of two ing in the covalent attachment of ubiquitin on to a substrate lobes—a larger N-terminal lobe responsible for recruit- via an isopeptide bond. In contrast to the RING (Really ing an E2 ~ ubiquitin complex, and a smaller C-terminal lobe that contains the catalytic cysteine required to cova- lently attach ubiquitin to its substrates (Lorenz 2018). The Steven A. Beasley and Roela Bardhi have contributed equally to HECT E3 ubiquitin ligase family can be further categorized this manuscript. into subfamilies, which includes the most well-examined NEDD4 subfamily. The NEDD4 HECT E3 ubiquitin ligases * Donald E. Spratt 2+ are comprised of a Ca -binding C2 domain, WW domains dspratt@clarku.edu that bind the PPxY protein–protein interaction motif, as well Gustaf H. Carlson School of Chemistry and Biochemistry, as the HECT domain required for its ubiquitylation activity. Clark University, 950 Main St., Worcester, MA 01610, USA Vol.:(0123456789) 1 3 16 S. A. Beasley et al. The 3D structures of several NEDD4 HECT domains family (Newark, CA, USA) and cloned into an ampicillin resist- members have been solved by X-ray crystallography, either ant T7-inducible vector with an N-terminal polyhistidine alone or complexed with E2 and/or ubiquitin (Lorenz 2018), tag (His -tag) followed by a TEV protease cleavage site however, there are many unanswered questions regarding (ENLYFQ). The His -TEV-ITCH C-terminal lobe con- how these enzymes assemble into multi-protein complexes struct was transformed into E. coli BL21(DE3) RIL+ and to synthesize polyubiquitin chains. grown at 37 °C in M9 media (2 × 1 L) supplemented with 15 13 ITCH, also known as Atrophin-1-interacting protein 4, NH Cl (1 g/L), C -glucose (2 g/L), 100 mg/L ampicillin 4 6 is a prominent member of the NEDD4 subfamily that regu- and 34 mg/L chloramphenicol. When the culture reached an lates signaling pathways involved in immune-cell differen- OD600 of 0.8, the cultures were induced with 1 mM IPTG at tiation, control of the inflammatory signaling pathways, and 16 °C for 20 h. The cells were harvested by centrifugation at apoptosis (Aki et al. 2015). ITCH is an intriguing HECT E3 6000×g for 10 min using a Sorvall LYNX 4000 superspeed ubiquitin ligase due to its ability to synthesize different poly - centrifuge with a Fiberlite F 10 − 4 × 1000 LEX Carbon ubiquitin lysine linkages (i.e. K29, K48, and/or K63-linked) Fiber rotor (Thermo-Fisher). Cell pellets were resuspended depending on the substrate it modifies, and this ubiquitin in 20 mL of wash buffer (50 mM Na HPO pH 8.0, 300 mM 2 4 linkage specificity is dependent on the last 60 amino acids NaCl, 10 mM imidazole) with 1 mM PMSF and an EDTA- of the C-terminal lobe of ITCH (Kim and Huibregtse 2009). free protease inhibitor mini tablet (Pierce), lysed using an For example, ITCH has been observed to build K29-linked Emulsiflex-C5 homogenizer (Avestin, Ottawa, ON, Canada), polyubiquitin chains on the transmembrane receptor protein and clarified by ultracentrifugation using a Optima L-80 Notch that leads to Notch undergoing endocytosis and lyso- XP ultracentrifuge with a 70.1 Ti rotor (Beckman-Coulter) somal degradation (Chastagner et al. 2008). ITCH knockout at 41,000 rpm for 40 min. The clarified supernatant was mice display an ‘Itchy’ phenotype caused by inflammatory then applied to 5 mL of HisPur Ni–NTA resin (Thermo- dysregulation due to unfettered Notch signaling (Chast- Fisher) pre-equilibrated with wash buffer at a flow rate of agner et al. 2008; Matesic et al. 2008). ITCH negatively 0.5 mL/min. After the resin was washed with 25 column regulates the Hippo tumor suppressor pathway by building volumes of wash buffer, the protein was eluted with 20 mL a K48-linked polyubiquitin chain on of the serine/threonine of elution buffer (50 mM Na HPO pH 8.0, 300 mM NaCl, 2 4 kinase LATS1, a tumor growth inhibitor that induces G2-M 250 mM imidazole). Fractions containing eluted protein arrest and apoptosis (Salah et al. 2014). ITCH also controls were pooled and incubated with recombinant TEV protease intracellular concentrations of p63 and p73, members of the for 1 h at 25 °C (1 mg TEV/50 mg eluted protein) to cleave tumor suppressor protein family, by K48-polyubiquitylation the His -tag, then dialyzed against wash buffer stirring over - to target these oncoproteins for proteosomal degradation night at 4 °C. The TEV cleaved protein was then reapplied (Melino et  al. 2008). K63-linked ITCH-dependent poly- to 5 mL of HisPur Ni–NTA resin at a flow rate of 0.5 mL/ 13 15 ubiquitination has also been observed for the transcription min and the flowthrough containing C– N-labeled ITCH factor p45/NF-E2 causing it to migrate out of the nucleus C-terminal lobe was collected and pooled. The protein was into the cytoplasm and thus inactivating p45/NF-E2 (Lee then concentrated using a Amicon 15 mL centrifugal filter et al. 2008). with a 10 kDa MWCO (Millipore) and loaded onto a HiLoad Presently there is no structural rationale for how ITCH 16/60 Superdex75 column equilibrated with 20 mM MES, is capable of building these different polyubiquitin chain 120  mM NaCl, 1  mM EDTA, 2  mM TCEP, pH 6.0  at a types and the catalytic mechanism for ITCH is currently flow rate of 1 mL/min using an ÄKTA pure 25L FPLC (GE unknown. Here we report the complete backbone and side- Healthcare Life Sciences). Fractions containing the puri- 1 13 15 13 15 chain H, C, and N resonance assignments for the cata- fied C– N-labeled ITCH C-terminal lobe, as assessed by lytic C-terminal lobe of ITCH (residues 784–903) using 3D SDS-PAGE, were pooled and concentrated. The resulting heteronuclear NMR spectroscopy. These resonance assign- ITCH C-terminal lobe protein had an additional “GS” at its ments will enable future structural and mechanistic studies N-terminus as a result of its cloning and TEV cleavage. After to better understand ITCH-dependent ubiquitylation. purification, the concentration of the protein was determined using the Bradford assay (Bio-Rad) or a A using a Nan- odrop One C UV/Vis spectrophotometer (Thermo-Fisher). Methods and experiments NMR spectroscopy Protein expression and purification The NMR samples used for resonance assignment of 13 15 The human HECT C-terminal lobe of ITCH (Uniprot: C– N-labeled human ITCH C-terminal lobe were pre- Q96J02, residues 784–903) with C835S and C855S substi- pared in 20 mM MES, 120 mM NaCl, 1 mM EDTA, 2 mM tutions was synthesized and codon-optimized by DNA2.0 TCEP, 10% DO/90% H O at pH 6.0. The samples were 2 2 1 3 1 13 15 H, C, and  N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin… 17 concentrated to a final volume of 600 μL and transferred to due to fast amide exchange with the solvent and/or high flex- a 5 mm O.D. thin walled NMR tube (New-Era). Imidazole ibility of the region. There were no unassigned peaks vis- 1 15 (1.6 mM) was added as an internal pH indicator to monitor ible on the well-dispersed H– N HSQC spectrum (Fig. 1). the pH of the sample during data acquisition (Baryshnikova One residue of particular interest that will be followed in et al. 2008). future chemical shift perturbation experiments is the cata- All NMR data were collected at 25 °C using a Varian lytic cysteine of ITCH (C871) as it has distinct Cβ peaks on 1 13 Inova 600 MHz 4-channel solution-state NMR Spectrometer the H– C HSQC whose chemical shift indicates that it is equipped with a 5-mm PFG triple-resonance probe housed reduced (Kornhaber et al. 2006) as would be required for its and maintained in the Sackler Sciences Center at Clark ubiquitylation activity. University. Backbone assignments and aliphatic side chain ITCH is a member of the NEDD4 subfamily of HECT assignments were determined using the following standard E3 ubiquitin ligases that shows a high degree of sequence 1 15 pulse sequences in the Varian Biopack in VnmrJ 3.0: H– N conservation (Fig. 2a) with many of these conserved resi- 1 13 HSQC, aliphatic H– C HSQC, HNCO, HN(CA)CO, dues found within the hydrophobic core of the protein. For HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH, ali- example, there are exceptional upfield peaks of the W813 1 15 1 15 phatic HCCH-TOCSY, C(CO)NH, H(CCO)NH, and H– N aromatic side chain in both the H– N HSQC and aromatic 1 13 NOESY. Aromatic sidechain assignments were determined H– C HSQC, likely due to shielding effect within the using an aromatic C-HSQC, HBCBCGCDHD and HBCB- hydrophobic core surrounded by three proximal aromatic CGCDCEHE experiments in combination with an aromatic residues (i.e. W793, Y799, and F812; Fig. 2b). Intriguingly, HCCH-TOCSY and aromatic C-NOESY experiments. All there is one exceptional tryptophan (W864) on the surface data were processed using NMRPipe and NMRDraw (Dela- that is found only in ITCH, as well as WWP1 (W883) and glio et al. 1995) and analyzed using NMRViewJ (Johnson WWP2 (W823). This residue, which resides on the same and Blevins 1994). All of the relevant peak lists and the face of the protein as the catalytic cysteine, deserves further 1 13 15 complete H, C, and N backbone and sidechain chemical attention as the mechanism of ubiquitin chain building and shift assignments have been deposited into the Biological lysine-linkage specificity depends on these subtle differences Magnetic Resonance Databank (http://www.bmrb.wisc.edu) between the different NEDD4 subfamily members. The sec- under ascension code 27477. ondary structure based on the chemical shift index (CSI) 3.0 web server analysis of chemical shifts (Hafsa et al. 2015) is Assignments and data deposition in good agreement with the known PDB structures of ITCH (Zhang et al. 2016) and other HECT domain C-terminal The non-artifact and non-proline amide protons (113/115; lobes with an αβα β α organization indicating that the pro- 2 3 98.26%), backbone atoms (350/360; 97.22%) and H chemi- tein is well folded and in the correct structural conformation cal shift assignments of the Hα (119/120, 99.17%) and Hβ (Fig. 3). (201/203, 99.01%) were definitively assigned. Every Cα In conclusion, we present the complete backbone and side and Cβ resonance were assigned except for the N-terminal chain resonance assignments of the catalytic HECT C-ter- artifact glycine and P878 (120/122, 98.36%). Aliphatic minal lobe of ITCH. These resonance assignments will be sidechain resonances of Cγ (60/62, 96.77%), Cδ (29/30, used to examine the role of inherent conformational flex- 96.67%), and Cε (13/13, 100%) were also assigned. Almost ibility within the C-terminal lobe of ITCH that will help us all nitrogen atoms—excluding the lysine, arginine, and his- decipher how ITCH is capable of building K29, K48, and tidine sidechain and proline backbone nitrogen atoms—were K63-linked polyubiquitin chains on its diverse intracellular definitively assigned (130/132; 98.48%). The missing amide substrates. peaks for K861 and W864 could not be assigned possibly 1 3 18 S. A. Beasley et al. 100.0 105.0 110.0 115.0 120.0 125.0 130.0 10.0 9.0 8.0 7.0 6.0 H Chemical Shift (ppm) 1 15 Fig. 1 Assigned observable H– N HSQC spectrum of the human 6.0, 120 mM NaCl, 1 mM EDTA, and 10% D O. Data was collected HECT C-terminal lobe of ITCH (residues 784–903, 2.6  mM). The on a Varian Inova 600-MHz NMR spectrometer at 25 °C. Peaks cor- spectrum is labeled according to the one-amino acid code and resi- responding to asparagine and glutamine side chain amides are con- due number of the human ITCH sequence. The NMR sample con- nected with a horizontal line 13 15 tained C and N-isotopically enriched ITCH in 20  mM MES pH 1 3 N Chemical Shift (ppm) 1 13 15 H, C, and  N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin… 19 AB * * ** *** * * * ******** ** ITCH/784-849 GMQEIDLNDWQRHAIYRH-YARTSKQIMWFWQFVKEIDNEKRMRLLQFVTGTCRLPVGGFADLMGSN F812 NEDD4/1198-1264 GLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAELYGSN NED4L/854-920 GLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSN HECW1/1486-1552 GTAEIDLNDWRNNTEYRGGYHDGHLVIRWFWAAVERFNNEQRLRLLQFVTGTSSVPYEGFAALRGSN HECW2/1452-1518 GTAEIDLSDWRNNTEYRGGYHDNHIVIRWFWAAVERFNNEQRLRLLQFVTGTSSIPYEGFASLRGSN W864 WWP1/803-868 GMQEVDLADWQRNTVYRH-YTRNSKQIIWFWQFVKETDNEVRMRLLQFVTGTCRLPLGGFAELMGSN WWP2/751-808 GMQEIDMSDWQKSTIYRH-YTKNSKQIQWFWQVVKEMDNEKRIRLLQFVTGTCRLPVGGFAELIGSN C-terminus SMURF1/634-699 GLDKIDLNDWKSNTRLKHCVAD-SNIVRWFWQAVETFDEERRARLLQFVTGSTRVPLQGFKALQGST W813 SMURF2/628-689 GLGKIDVNDWKVNTRLKHCTPD-SNIVKWFWKAVEFFDEERRARLLQFVTGSSRVPLQGFKALQ--- Y799 * ** ****** * *** * ** * * * * ITCH/850-903 ---GPQKFCIEKVGK-ENWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGF-GQE N-terminus NEDD4/1265-1319 ---GPQSFTVEQWGT-PEKLPRAHTCFNRLDLPPYESFEELWDKLQMAIENTQGFDGVD NED4L/921-975 ---GPQLFTIEQWGS-PEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVD HECW1/1553-1606 ---GLRRFCIEKWGK-ITSLPRAHTCFNRLDLPPYPSYSMLYEKLLTAVEETSTF-GLE W793 C871 HECW2/1519-1572 ---GPRRFCVEKWGK-ITALPRAHTCFNRLDLPPYPSFSMLYEKLLTAVEETSTF-GLE WWP1/869-922 ---GPQKFCIEKVGK-DTWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGF-GQE WWP2/809-870 ---GPQKFCIDKVGK-ETWLPRSHTCFNRLDLPPYKSYEQLREKLLYAIEETEGF-GQE SMURF1/700-757 GAAGPRLFTIHLIDANTDNLPKAHTCFNRIDIPPYESYEKLYEKLLTAVEETCGF-AVE SMURF2/690-748 GAAGPRLFTIHQIDACTNNLPKAHTCFNRIDIPPYESYEKLYEKLLTAIEETCGF-AVE Fig. 2 a Multiple sequence alignment of the C-terminal lobes of the are marked with an asterisk. b Structure of ITCH C-terminal lobe NEDD4 HECT E3 ubiquitin ligase subfamily. The sequence align- (PDB:3TUG) highlighting the catalytic cysteine C871 (red), as well ment was performed using T-Coffee (Notredame et al. 2000) followed as the W813 (cyan), surrounded by aromatic residues W793, Y799, by manual curation in Jalview (Waterhouse et al. 2009). The α-helices and F812 (yellow). The solvent exposed tryptophan W864 is shown and β-sheets based on the crystal structure of ITCH are shown as in magenta. Noteworthy residues discussed in the text are also high- cylinders and arrows, respectively. Absolutely conserved residues lighted in panel A using the same color scheme Fig. 3 Predicted secondary 1.0 structural regions of the ITCH C-terminal lobe. The probabil- ity plot was made by inputting the experimentally determined 0.8 resonance assignments for ITCH into the online webserver CSI 3.0 (Hafsa et al. 2015). The 0.6 propensity to form an a-helix or b-sheet are denoted in red and blue, respectively 0.4 0.2 791 801 811 821 831 841 851 861 871 881 891901 Residue Acknowledgements The authors would like to thank Dr. Guoxing Lin References for his assistance in setting up experiments and for maintaining the 600 MHz NMR spectrometer housed in the Carlson School of Chem- Aki D, Zhang W, Liu YC (2015) The E3 ligase Itch in immune regula- istry and Biochemistry at Clark University. This work was supported tion and beyond. Immunol Rev 266:6–26. https://doi.or g/10.1111/ by a grant from the National Institutes of Health (R15GM126432) and imr.12301 start-up funds from Clark University. Baryshnikova OK, Williams TC, Sykes BD (2008) Internal pH indica- tors for biomolecular. NMR J Biomol NMR 41:5–7. https ://doi. 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