Crystal structure, biochemical and biophysical characterisation of NHR1 domain of E3 Ubiquitin ligase neutralized


Notch signaling controls diverse developmental decisions of central importance to cell activity. One of the conserved positive regulators of Notch signaling is Neuralized, the E3 Ubiquitin ligase enzyme that regulates signaling activity by endocytosis. Neuralized has two novel repeats, NHR1 and NHR2, with a RING finger motif at the C-terminus. Both endocytosis of the Notch ligand, Delta, and inhibition of Notch signaling by Tom, a bearded family member, require the NHR1 domain. Here we describe the first crystal structure of NHR1 domain from Drosophila melanogaster, solved to 2.1 A resolution by X-ray analysis. Using NMR and other biophysical techniques we define a minimal binding region of Tom, consisting of 12 residues, which interacts with NHR1 and show by interfacial analysis of protein monolayers that NHR1 binds PI4P. Taken together, the studies provide insight into molecular interactions that are important for Notch signaling.

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Gupta, D. , Beaufils, S. , Vie, V. , Paboeuf, G. , Broadhurst, B. , Schweisguth, F. , L. Blundell, T. and M. Bolanos-Garcia, V. (2013) Crystal structure, biochemical and biophysical characterisation of NHR1 domain of E3 Ubiquitin ligase neutralized. Advances in Enzyme Research, 1, 61-75. doi: 10.4236/aer.2013.13007.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Pavlopoulos, E., et al. (2001) Neuralized encodes a peripheral membrane protein involved in delta signaling and endocytosis. Developmental Cell, 1, 807-816. doi:10.1016/S1534-5807(01)00093-4
[2] Liu, S., et al. (2012) Functional analysis of the NHR2 domain indicates that oli-gomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling. Molecular Cell Biology, 32, 4933-4945. doi:10.1128/MCB.00711-12
[3] Le Borgne, R., et al. (2005) Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biology, 3, e96. doi:10.1371/journal.pbio.0030096
[4] Lai, E.C., et al. (2001) Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta. Developmental Cell, 1, 783-794. doi:10.1016/S1534-5807(01)00092-2
[5] Yeh, E., et al. (2001) Neuralized functions as an E3 ubiquitin ligase during Drosophila development. Current Biology, 11, 1675-1679. doi:10.1016/S0960-9822(01)00527-9
[6] Lai, E.C., et al. (2000) Antagonism of notch signaling activity by members of a novel protein family encoded by the bearded and enhancer of split gene complexes. Development, 127,291-306.
[7] Bardin, A.J., and Schweisguth, F. (2006) Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila. Developmental Cell, 10, 245-255. doi:10.1016/j.devcel.2005.12.017
[8] He, F., et al. (2009) Structural and Functional Characterization of the NHR1 Domain of the Drosophila Neuralized E3 Ligase in the Notch Signaling Pathway. Journal of Molecular Biology, 393, 478-495. doi:10.1016/j.jmb.2009.08.020
[9] Skwarek, L.C., et al. (2007) Neuralized contains a phosphoinositide-binding motif required downstream of ubiquitination for delta endocytosis and notch signaling. Developmental Cell, 13, 783-795. doi:10.1016/j.devcel.2007.10.020
[10] Weinmaster, G. and Fischer, J.A. (2011) Notch ligand ubiquitylation: What is it good for? Developmental Cell, 21, 134-144. doi:10.1016/j.devcel.2011.06.006
[11] Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology, 276, 307-326. doi:10.1016/S0076-6879(97)76066-X
[12] CCP4. (1994) Collaborative Computational Project. Acta Crystallographica Section D: Biological Crystallography, D50, 760.
[13] Navaza, J. (1994) AMoRe: an automated package for molecular replacement. Acta Crystallographica Section A: Foundations of Crystallography, A50, 157-163. doi:10.1107/S0108767393007597
[14] Murshudova, G.N., Vagin, A.A. and Dodson, E.J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D: Biological Crystallography, D53, 240-255. doi:10.1107/S0907444996012255
[15] Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography, D60, 2126-2132. doi:10.1107/S0907444904019158
[16] Laskowski, R.A., et al. (1993) PROCHECK—A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283-291. doi:10.1107/S0021889892009944
[17] Baker, N.A., et al. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proceedings of the National Academy of Science USA, 98, 10037-10041. (APBS)
[18] Sali, A. and Blundell, T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology, 234,779-815. doi:10.1006/jmbi.1993.1626
[19] Lo, M.C., et al. (2004) Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Analytical Biochemistry, 332, 153-159. doi:10.1016/j.ab.2004.04.031
[20] Azzam, R.M.A. and N.M. Bashara. (1977) Ellipsometry and polarized light. In: Cowley, J.M. Ed., North Holland Personal Library, North Holland Personal Library, Amsterdam, 340.
[21] De Feijter, J.A., Benjamins, J. and Veer, F.A. (1978) Ellipsometry as a tool to study the adsorption behaviour of synthetic and biopolymers at the air-water interface. Biopolymers, 17, 1759-1772. doi:10.1002/bip.1978.360170711
[22] Venien-Bryan, C., et al. (1998) Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy. Biophysical Journal, 74, 2649-2657. doi:10.1016/S0006-3495(98)77970-6
[23] Renault, A., et al. (1999) Surface-induced polymerization of actin. Biophysical Journal, 76, 1580-1590. doi:10.1016/S0006-3495(99)77317-0
[24] Bolanos-Garcia, V.M., et al. (2005) The conserved N-terminal region of the mitotic checkpoint protein BUBR1: A putative TPR motif of high surface activity. Biophysical Journal, 89, 2640-2649. doi:10.1529/biophysj.105.063511
[25] Shi, J., Blundell, T.L. and Mizuguchi, K. (2001) FUGUE: Sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. Journal of Molecular Biology, 310, 243-257. doi:10.1006/jmbi.2001.4762
[26] Linossi, E.M. and Nicholson, S.E. (2012) The SOCS box-adapting proteins for ubiquitination and proteasomal degradation. IUBMB Life, 64, 316-323. doi:10.1002/iub.1011
[27] Mizuguchi, K., et al. (1998) JOY: Protein sequence-structure representation and analysis. Bioin-formatics, 14, 617- 623. doi:10.1093/bioinformatics/14.7.617
[28] Woo, J.S., et al. (2006) Structural basis for protein recognition by B30.2/SPRY domains. Molecular Cell, 24, 967-976. doi:10.1016/j.molcel.2006.11.009
[29] Ponting, C.P., et al. (2001) Novel protein domains and repeats in Drosophila melanogaster: Insights into structure, function, and evolution. Genome Research, 11, 1996-2008. doi:10.1101/gr.198701
[30] Commisso, C. and Boulianne, G.L. (2007) The NHR1 domain of Neuralized binds Delta and mediates Delta trafficking and Notch signaling. Molecular Biology of the Cell, 18, 1-13. doi:10.1091/mbc.E06-08-0753
[31] Commisso, C. and Boulianne, G.L. (2008) The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-depen-dent inhibition of nuclear import. Journal of Molecular Biology, 375, 1125-1140. doi:10.1016/j.jmb.2007.11.043
[32] Innis, C.A., Shi, J. and Blundell, T.L. (2000) Evolutionary trace analysis of TGF-beta and related growth factors: Implications for site-directed muta-genesis. Protein Engineering, 13, 839-847. doi:10.1093/protein/13.12.839
[33] Bickerton, G.R., Higueruelo, A.P. and Blundell, T.L. (2011) Comprehensive, atomic-level characterization of structurally characterized protein-protein interactions: The PICCOLO database. BMC Bioinformatics, 12, 313. doi:10.1186/1471-2105-12-313
[34] Gupta D., (2010) Structural and functional study on Notch signaling pathway and its regulatory proteins. PhD Thesis, University of Cambridge, England.
[35] Glittenberg, M., et al. (2006) Role of conserved intracellular motifs in serrate signaling, cis-inhibition and endocytosis. EMBO Journal, 25, 4697-4706. doi:10.1038/sj.emboj.7601337
[36] Damodaran, S.A.C.S.R. (2001) Molecular basis for protein adsorption at fluid-fluid interfaces. In: Dickinson, E., Miller, R., Damodaran S. and Rao, C.S., Eds., Food Colloids: Fundamental of Formulation, Royal Society of Chemistry, London, 165-180.
[37] Beaufils, S., et al. (2008) Characterization of the tetratricopeptide-containing domain of BUB1, BUBR1, and PP5 proves that domain amphiphilicity over amino acid sequence specificity governs protein adsorption and interfacial activity. Journal of Physical Chemistry B, 112, 7984-7991. doi:10.1021/jp711222s
[38] Lee, S., et al. (2012) Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases. Journal of Biological Chemistry, 287, 5988-6001.

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