It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region.

Central European Institute of Technology Masaryk University Brno Czech Republic

Maurice Wohl Clinical Neuroscience Institute Institute of Psychiatry Psychology and Neuroscience King's College London London United Kingdom

Maurice Wohl Clinical Neuroscience Institute Institute of Psychiatry Psychology and Neuroscience King's College London London United Kingdom; Department of Molecular Medicine University of Pavia Pavia Italy

Medical Research Council Biomolecular NMR Centre The Francis Crick Institute London United Kingdom

Zobrazit více v PubMed

Paulson H.L., Perez M.K., Pittman R.N. Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron. 1997;19:333–344. PubMed

Ellisdon A.M., Thomas B., Bottomley S.P. The two-stage pathway of ataxin-3 fibrillogenesis involves a polyglutamine-independent step. J. Biol. Chem. 2006;281:16888–16896. PubMed

Saunders H.M., Gilis D., Bottomley S.P. Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein. Protein Sci. 2011;20:1675–1681. PubMed PMC

Doss-Pepe E.W., Stenroos E.S., Madura K. Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis. Mol. Cell. Biol. 2003;23:6469–6483. PubMed PMC

Scaglione K.M., Zavodszky E., Paulson H.L. Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP. Mol. Cell. 2011;43:599–612. PubMed PMC

Tu Y., Liu H., Tang T.S. Ataxin-3 promotes genome integrity by stabilizing Chk1. Nucleic Acids Res. 2017;45:4532–4549. PubMed PMC

Chatterjee A., Saha S., Hazra T.K. The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3′-phosphatase in spinocerebellar ataxia type 3 pathogenesis. PLoS Genet. 2015;11:e1004749. PubMed PMC

Jana N.R., Dikshit P., Nukina N. Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes. J. Biol. Chem. 2005;280:11635–11640. PubMed

Bonanomi M., Mazzucchelli S., Tortora P. Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome. Int. J. Biochem. Cell Biol. 2014;51:58–64. PubMed

Ferro A., Carvalho A.L., Maciel P. NEDD8: a new ataxin-3 interactor. Biochim. Biophys. Acta. 2007;1773:1619–1627. PubMed

Moir D., Stewart S.E., Botstein D. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics. 1982;100:547–563. PubMed PMC

Meyer H.H., Wang Y., Warren G. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 2002;21:5645–5652. PubMed PMC

Finkelstein A.V. 50+ years of protein folding. Biochemistry (Mosc.) 2018;83(Suppl 1):S3–S18. PubMed

DeForte S., Uversky V.N. Order, disorder, and everything in between. Molecules. 2016;21::E1090. PubMed PMC

Masino L., Musi V., Pastore A. Domain architecture of the polyglutamine protein ataxin-3: a globular domain followed by a flexible tail. FEBS Lett. 2003;549:21–25. PubMed

Nicastro G., Masino L., Pastore A. Josephin domain of ataxin-3 contains two distinct ubiquitin-binding sites. Biopolymers. 2009;91:1203–1214. PubMed

Song A.X., Zhou C.J., Hu H.Y. Structural transformation of the tandem ubiquitin-interacting motifs in ataxin-3 and their cooperative interactions with ubiquitin chains. PLoS One. 2010;5:e13202. PubMed PMC

Nicastro G., Menon R.P., Pastore A. The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition. Proc. Natl. Acad. Sci. USA. 2005;102:10493–10498. PubMed PMC

Tong K.I., Yamamoto M., Tanaka T. Selective isotope labeling of recombinant proteins in Escherichia coli. Methods Mol. Biol. 2012;896:439–448. PubMed

Motáčková V., Nováček J., Sklenář V. Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments. J. Biomol. NMR. 2010;48:169–177. PubMed PMC

Cavanagh J., Fairbrother W.J., Skelton N.J. Academic Press; Cambridge, MA: 2006. Protein NMR Spectroscopy.

Delaglio F., Grzesiek S., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 1995;6:277–293. PubMed

Kazimierczuk K., Koźmiński W., Zhukov I. Two-dimensional fourier transform of arbitrarily sampled NMR data sets. J. Magn. Reson. 2006;179:323–328. PubMed

Kazimierczuk K., Zawadzka A., Zhukov I. Random sampling of evolution time space and Fourier transform processing. J. Biomol. NMR. 2006;36:157–168. PubMed

Kazimierczuk K., Zawadzka A., Koźmiński W. Narrow peaks and high dimensionalities: exploiting the advantages of random sampling. J. Magn. Reson. 2009;197:219–228. PubMed

Vranken W.F., Boucher W., Laue E.D. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins. 2005;59:687–696. PubMed

Helmus J.J., Jaroniec C.P. Nmrglue: an open source Python package for the analysis of multidimensional NMR data. J. Biomol. NMR. 2013;55:355–367. PubMed PMC

Masino L., Nicastro G., Pastore A. The Josephin domain determines the morphological and mechanical properties of ataxin-3 fibrils. Biophys. J. 2011;100:2033–2042. PubMed PMC

Dubey A., Kadumuri R.V., Atreya H.S. Rapid NMR assignments of proteins by using optimized combinatorial selective unlabeling. ChemBioChem. 2016;17:334–340. PubMed

Lescop E., Kern T., Brutscher B. Guidelines for the use of band-selective radiofrequency pulses in hetero-nuclear NMR: example of longitudinal-relaxation-enhanced BEST-type 1H-15N correlation experiments. J. Magn. Reson. 2010;203:190–198. PubMed

MacRaild C.A., Zachrdla M., Norton R.S. Conformational dynamics and antigenicity in the disordered malaria antigen merozoite surface protein 2. PLoS One. 2015;10:e0119899. PubMed PMC

Nyarko A., Song Y., Barbar E. Multiple recognition motifs in nucleoporin Nup159 provide a stable and rigid Nup159-Dyn2 assembly. J. Biol. Chem. 2013;288:2614–2622. PubMed PMC

Orbán-Németh Z., Henen M.A., Konrat R. Backbone and partial side chain assignment of the microtubule binding domain of the MAP1B light chain. Biomol. NMR Assign. 2014;8:123–127. PubMed PMC

Solyom Z., Schwarten M., Brutscher B. BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J. Biomol. NMR. 2013;55:311–321. PubMed

Marsh J.A., Singh V.K., Forman-Kay J.D. Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. Protein Sci. 2006;15:2795–2804. PubMed PMC

Wishart D.S., Sykes B.D., Richards F.M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992;31:1647–1651. PubMed

Li P., Huey-Tubman K.E., Bjorkman P.J. The structure of a polyQ-anti-polyQ complex reveals binding according to a linear lattice model. Nat. Struct. Mol. Biol. 2007;14:381–387. PubMed

Masino L., Kelly G., Pastore A. Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins. FEBS Lett. 2002;513:267–272. PubMed

Maciejewski M.W., Liu D., Mullen G.P. Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity. J. Mol. Biol. 2000;296:229–253. PubMed

Burnett B., Li F., Pittman R.N. The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity. Hum. Mol. Genet. 2003;12:3195–3205. PubMed

Chai Y., Berke S.S., Paulson H.L. Poly-ubiquitin binding by the polyglutamine disease protein ataxin-3 links its normal function to protein surveillance pathways. J. Biol. Chem. 2004;279:3605–3611. PubMed

Chow M.K., Mackay J.P., Bottomley S.P. Structural and functional analysis of the Josephin domain of the polyglutamine protein ataxin-3. Biochem. Biophys. Res. Commun. 2004;322:387–394. PubMed

Chow M.K., Paulson H.L., Bottomley S.P. Destabilization of a non-pathological variant of ataxin-3 results in fibrillogenesis via a partially folded intermediate: a model for misfolding in polyglutamine disease. J. Mol. Biol. 2004;335:333–341. PubMed

Nicastro G., Habeck M., Pastore A. Structure validation of the Josephin domain of ataxin-3: conclusive evidence for an open conformation. J. Biomol. NMR. 2006;36:267–277. PubMed

Bermel W., Felli I.C., Zawadzka-Kazimierczuk A. High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins. J. Biomol. NMR. 2013;57:353–361. PubMed

O’Hare B., Benesi A.J., Showalter S.A. Incorporating 1H chemical shift determination into 13C-direct detected spectroscopy of intrinsically disordered proteins in solution. J. Magn. Reson. 2009;200:354–358. PubMed

Sahu D., Bastidas M., Showalter S.A. Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods. Anal. Biochem. 2014;449:17–25. PubMed PMC

Lawrence C.W., Showalter S.A. Carbon-detected (15)N NMR spin relaxation of an intrinsically disordered protein: FCP1 dynamics unbound and in complex with RAP74. J. Phys. Chem. Lett. 2012;3:1409–1413. PubMed

Davies H.A., Rigden D.J., Madine J. Probing medin monomer structure and its amyloid nucleation using 13C-direct detection NMR in combination with structural bioinformatics. Sci. Rep. 2017;7:45224. PubMed PMC

Nicastro G., Masino L., Pastore A. Assignment of the 1H, 13C, and 15N resonances of the Josephin domain of human ataxin-3. J. Biomol. NMR. 2004;30:457–458. PubMed

Eftekharzadeh B., Piai A., Salvatella X. Sequence context influences the structure and aggregation behavior of a PolyQ tract. Biophys. J. 2016;110:2361–2366. PubMed PMC

Baias M., Smith P.E., Frydman L. Structure and dynamics of the huntingtin exon-1 N-terminus: A solution NMR perspective. J. Am. Chem. Soc. 2017;139:1168–1176. PubMed

Thakur A.K., Jayaraman M., Wetzel R. Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism. Nat. Struct. Mol. Biol. 2009;16:380–389. PubMed PMC

Kim M.W., Chelliah Y., Bezprozvanny I. Secondary structure of huntingtin amino-terminal region. Structure. 2009;17:1205–1212. PubMed PMC

Legleiter J., Lotz G.P., Muchowski P.J. Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment. J. Biol. Chem. 2009;284:21647–21658. PubMed PMC

Daldin M., Fodale V., Caricasole A. Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models. Sci. Rep. 2017;7:5070. PubMed PMC

Bennett M.J., Huey-Tubman K.E., Bjorkman P.J. A linear lattice model for polyglutamine in CAG-expansion diseases. Proc. Natl. Acad. Sci. USA. 2002;99:11634–11639. PubMed PMC

Persichetti F., Trettel F., MacDonald M.E. Mutant huntingtin forms in vivo complexes with distinct context-dependent conformations of the polyglutamine segment. Neurobiol. Dis. 1999;6:364–375. PubMed

Miller J., Arrasate M., Finkbeiner S. Identifying polyglutamine protein species in situ that best predict neurodegeneration. Nat. Chem. Biol. 2011;7:925–934. PubMed PMC

Trottier Y., Lutz Y., Tora L. Polyglutamine expansion as a pathological epitope in Huntington’s disease and four dominant cerebellar ataxias. Nature. 1995;378:403–406. PubMed

Zhemkov V.A., Kulminskaya A.A., Kim M. The 2.2-Angstrom resolution crystal structure of the carboxy-terminal region of ataxin-3. FEBS Open Bio. 2016;6:168–178. PubMed PMC

Bhattacharyya A., Thakur A.K., Wetzel R. Oligoproline effects on polyglutamine conformation and aggregation. J. Mol. Biol. 2006;355:524–535. PubMed

Darnell G., Orgel J.P., Meredith S.C. Flanking polyproline sequences inhibit beta-sheet structure in polyglutamine segments by inducing PPII-like helix structure. J. Mol. Biol. 2007;374:688–704. PubMed

Shen K., Calamini B., Frydman J. Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract. eLife. 2016;5:e18065. PubMed PMC

Totzeck F., Andrade-Navarro M.A., Mier P. The protein structure Context of PolyQ Regions. PLoS One. 2017;12:e0170801. PubMed PMC

Crick S.L., Ruff K.M., Pappu R.V. Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. USA. 2013;110:20075–20080. PubMed PMC

Nagai Y., Inui T., Toda T. A toxic monomeric conformer of the polyglutamine protein. Nat. Struct. Mol. Biol. 2007;14:332–340. PubMed

Peters-Libeu C., Miller J., Finkbeiner S. Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure. J. Mol. Biol. 2012;421:587–600. PubMed PMC

Klein F.A., Zeder-Lutz G., Trottier Y. Linear and extended: a common polyglutamine conformation recognized by the three antibodies MW1, 1C2 and 3B5H10. Hum. Mol. Genet. 2013;22:4215–4223. PubMed

Hoop C.L., Lin H.K., van der Wel P.C. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core. Proc. Natl. Acad. Sci. USA. 2016;113:1546–1551. PubMed PMC

Schneider R., Schumacher M.C., Baldus M. Structural characterization of polyglutamine fibrils by solid-state NMR spectroscopy. J. Mol. Biol. 2011;412:121–136. PubMed

Rao M.V., Williams D.R., Loll P.J. Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding. J. Biol. Chem. 2017;292:18392–18407. PubMed PMC