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Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations

  • Marie Ménade
    Footnotes
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Guennadi Kozlov
    Footnotes
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Jean-François Trempe
    Footnotes
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Harshit Pande
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Solomon Shenker
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Sihara Wickremasinghe
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Xinlu Li
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Hamed Hojjat
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Marie-Josée Dicaire
    Affiliations
    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
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  • Bernard Brais
    Affiliations
    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
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  • Peter S. McPherson
    Affiliations
    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
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  • Michael J.H. Wong
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Jason C. Young
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Kalle Gehring
    Correspondence
    To whom correspondence should be addressed:Dept. of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Rm. 473, Montreal, Quebec H3G 0B1, Canada. Tel.:514-398-7287;
    Affiliations
    Department of Biochemistry, McGill Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Author Footnotes
    1 These authors contributed equally to this work.
    2 Present address: Dept. of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada.
    4 The abbreviations used are: ARSACSautosomal recessive spastic ataxia of Charlevoix-SaguenayUblubiquitin-like domainSIRPTsacsin internal repeatHEPNhigher eukaryotes and prokaryotes nucleotide-bindingNBDnucleotide-binding domainGRP94glucose-regulated protein 94TRAP1tumor necrosis factor receptor–associated protein 1r.m.s.d.root mean square deviationPDBProtein Data Bank3-MetF270M/K291M/L318M4-MetI113M/F270M/K291M/L308MITCisothermal titration calorimetrySAXSsmall-angle X-ray scatteringRgradius of gyrationBicineN,N-bis(2-hydroxyethyl)glycineCHESSCornell High-Energy Synchrotron Source.
      Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein.

      Introduction

      Sacsin is a gigantic 520-kDa protein that is a product of the SACS gene, located on human chromosome 13 (13q12) (
      • Bouchard J.P.
      • Richter A.
      • Mathieu J.
      • Brunet D.
      • Hudson T.J.
      • Morgan K.
      • Melançon S.B.
      Autosomal recessive spastic ataxia of Charlevoix-Saguenay.
      ,
      • Engert J.C.
      • Bérubé P.
      • Mercier J.
      • Doré C.
      • Lepage P.
      • Ge B.
      • Bouchard J.P.
      • Mathieu J.
      • Melançon S.B.
      • Schalling M.
      • Lander E.S.
      • Morgan K.
      • Hudson T.J.
      • Richter A.
      ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF.
      ). Mutations in sacsin cause the neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)
      The abbreviations used are: ARSACS
      autosomal recessive spastic ataxia of Charlevoix-Saguenay
      Ubl
      ubiquitin-like domain
      SIRPT
      sacsin internal repeat
      HEPN
      higher eukaryotes and prokaryotes nucleotide-binding
      NBD
      nucleotide-binding domain
      GRP94
      glucose-regulated protein 94
      TRAP1
      tumor necrosis factor receptor–associated protein 1
      r.m.s.d.
      root mean square deviation
      PDB
      Protein Data Bank
      3-Met
      F270M/K291M/L318M
      4-Met
      I113M/F270M/K291M/L308M
      ITC
      isothermal titration calorimetry
      SAXS
      small-angle X-ray scattering
      Rg
      radius of gyration
      Bicine
      N,N-bis(2-hydroxyethyl)glycine
      CHESS
      Cornell High-Energy Synchrotron Source.
      first described in the Canadian province of Québec in 1978 (
      • Bouchard J.P.
      • Barbeau A.
      • Bouchard R.
      • Bouchard R.W.
      Autosomal recessive spastic ataxia of Charlevoix-Saguenay.
      ). Since then, cases have been reported worldwide with close to 200 mutations known (
      • Liu L.
      • Li X.B.
      • Zi X.H.
      • Shen L.
      • Hu Zh. M.
      • Huang Sh. X.
      • Yu D.L.
      • Li H.B.
      • Xia K.
      • Tang B.S.
      • Zhang R.X.
      A novel hemizygous SACS mutation identified by whole exome sequencing and SNP array analysis in a Chinese ARSACS patient.
      ,
      • Thiffault I.
      • Dicaire M.J.
      • Tetreault M.
      • Huang K.N.
      • Demers-Lamarche J.
      • Bernard G.
      • Duquette A.
      • Larivière R.
      • Gehring K.
      • Montpetit A.
      • McPherson P.S.
      • Richter A.
      • Montermini L.
      • Mercier J.
      • Mitchell G.A.
      • et al.
      Diversity of ARSACS mutations in French-Canadians.
      ). In the Quebec population, ARSACS is characterized by an early-onset progressive cerebellar atrophy, peripheral neuropathy, and thickening of the retina; clinical symptoms in most cases consist of cerebellar ataxia, spasticity, distal amyotrophy, nystagmus, and dysarthria, although the identification of new mutations has broadened its clinical spectrum (
      • Blumkin L.
      • Bradshaw T.
      • Michelson M.
      • Kopler T.
      • Dahari D.
      • Lerman-Sagie T.
      • Lev D.
      • Chapple J.P.
      • Leshinsky-Silver E.
      Molecular and functional studies of retinal degeneration as a clinical presentation of SACS-related disorder.
      ,
      • Larivière R.
      • Gaudet R.
      • Gentil B.J.
      • Girard M.
      • Conte T.C.
      • Minotti S.
      • Leclerc-Desaulniers K.
      • Gehring K.
      • McKinney R.A.
      • Shoubridge E.A.
      • McPherson P.S.
      • Durham H.D.
      • Brais B.
      Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.
      ). Sacsin is localized in the cytoplasm of neuronal cells in the soma, axons, and dendrites, and some is found in close proximity to mitochondria. Sacsin mRNA and its product are highly expressed in Purkinje cells (
      • Parfitt D.A.
      • Michael G.J.
      • Vermeulen E.G.
      • Prodromou N.V.
      • Webb T.R.
      • Gallo J.M.
      • Cheetham M.E.
      • Nicoll W.S.
      • Blatch G.L.
      • Chapple J.P.
      The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.
      ). ARSACS patient fibroblasts and neurons with sacsin knockdown present a hyperfused mitochondrial network with some accumulation of mitochondria in the soma and proximal dendrites (
      • Girard M.
      • Larivière R.
      • Parfitt D.A.
      • Deane E.C.
      • Gaudet R.
      • Nossova N.
      • Blondeau F.
      • Prenosil G.
      • Vermeulen E.G.
      • Duchen M.R.
      • Richter A.
      • Shoubridge E.A.
      • Gehring K.
      • McKinney R.A.
      • Brais B.
      • et al.
      Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).
      ). Recent studies in patient fibroblasts demonstrate that sacsin knockout increases reactive oxygen species production (
      • Bradshaw T.Y.
      • Romano L.E.
      • Duncan E.J.
      • Nethisinghe S.
      • Abeti R.
      • Michael G.J.
      • Giunti P.
      • Vermeer S.
      • Chapple J.P.
      A reduction in Drp1 mediated fission compromises mitochondrial health in autosomal recessive spastic ataxia of Charlevoix Saguenay.
      ,
      • Criscuolo C.
      • Procaccini C.
      • Meschini M.C.
      • Cianflone A.
      • Carbone R.
      • Doccini S.
      • Devos D.
      • Nesti C.
      • Vuillaume I.
      • Pellegrino M.
      • Filla A.
      • De Michele G.
      • Matarese G.
      • Santorelli F.M.
      Powerhouse failure and oxidative damage in autosomal recessive spastic ataxia of Charlevoix-Saguenay.
      ).
      Little is known about the mechanism of sacsin function. A study of sacsin chaperone activity showed some protective function against polyglutamine-expanded toxicity of ataxin-1. Namely, when sacsin was knocked down in cells using siRNA, the toxicity of the spinocerebellar ataxia type-1 ataxin-1[Q82] mutant protein was enhanced, whereas there was no toxicity with the ataxin-1[Q30] protein that contains a normal number of glutamine residues (
      • Parfitt D.A.
      • Michael G.J.
      • Vermeulen E.G.
      • Prodromou N.V.
      • Webb T.R.
      • Gallo J.M.
      • Cheetham M.E.
      • Nicoll W.S.
      • Blatch G.L.
      • Chapple J.P.
      The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.
      ). This suggests a possible role of sacsin in degradation of misfolded proteins. A firefly luciferase in vitro refolding experiment showed a weak activity with the first sacsin internal repeat, SIRPT1 (
      • Anderson J.F.
      • Siller E.
      • Barral J.M.
      The neurodegenerative-disease-related protein sacsin is a molecular chaperone.
      ).
      Structurally, sacsin is a multidomain protein (Fig. 1A) with an N-terminal ubiquitin-like (Ubl) domain that was shown to bind to the proteasome via proteasomal 20S α subunit C8 (
      • Parfitt D.A.
      • Michael G.J.
      • Vermeulen E.G.
      • Prodromou N.V.
      • Webb T.R.
      • Gallo J.M.
      • Cheetham M.E.
      • Nicoll W.S.
      • Blatch G.L.
      • Chapple J.P.
      The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.
      ). Sacsin has also been identified as a substrate of the E3 ubiquitin ligase Ube3A, the product of a gene associated with the neurological disorder Angelman syndrome (
      • Greer P.L.
      • Hanayama R.
      • Bloodgood B.L.
      • Mardinly A.R.
      • Lipton D.M.
      • Flavell S.W.
      • Kim T.K.
      • Griffith E.C.
      • Waldon Z.
      • Maehr R.
      • Ploegh H.L.
      • Chowdhury S.
      • Worley P.F.
      • Steen J.
      • Greenberg M.E.
      The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc.
      ). Partial sequence similarity of RAD23A(231–285), a binding partner of Ube3A, to residues 3660–3735 of sacsin led to the suggestion that a putative xeroderma pigmentosum complementation group C domain exists in SIRPT3 (
      • Greer P.L.
      • Hanayama R.
      • Bloodgood B.L.
      • Mardinly A.R.
      • Lipton D.M.
      • Flavell S.W.
      • Kim T.K.
      • Griffith E.C.
      • Waldon Z.
      • Maehr R.
      • Ploegh H.L.
      • Chowdhury S.
      • Worley P.F.
      • Steen J.
      • Greenberg M.E.
      The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc.
      ,
      • Kamionka M.
      • Feigon J.
      Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction.
      ). Toward the C terminus, sacsin contains a J domain immediately followed by a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain (
      • Parfitt D.A.
      • Michael G.J.
      • Vermeulen E.G.
      • Prodromou N.V.
      • Webb T.R.
      • Gallo J.M.
      • Cheetham M.E.
      • Nicoll W.S.
      • Blatch G.L.
      • Chapple J.P.
      The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.
      ). The J domain was demonstrated to be functional using a complementation assay technique with Hsp70 chaperone (
      • Parfitt D.A.
      • Michael G.J.
      • Vermeulen E.G.
      • Prodromou N.V.
      • Webb T.R.
      • Gallo J.M.
      • Cheetham M.E.
      • Nicoll W.S.
      • Blatch G.L.
      • Chapple J.P.
      The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.
      ). The sacsin J domain structure was previously determined by NMR (available as Protein Data Bank (PDB) code 1IUR). HEPN domains were originally identified in bacteria with a role in resistance to aminoglycosides antibiotics (
      • Matsumura M.
      • Katakura Y.
      • Imanaka T.
      • Aiba S.
      Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110.
      ); it was subsequently recognized by bioinformatics analysis that the HEPN domain exists in humans exclusively in sacsin (
      • Grynberg M.
      • Erlandsen H.
      • Godzik A.
      HEPN: a common domain in bacterial drug resistance and human neurodegenerative proteins.
      ). A high-resolution crystal structure of the HEPN revealed that it forms a stable dimer that mediates sacsin dimerization and binds GTP and other nucleotides with low-micromolar affinity (
      • Kozlov G.
      • Denisov A.Y.
      • Girard M.
      • Dicaire M.J.
      • Hamlin J.
      • McPherson P.S.
      • Brais B.
      • Gehring K.
      Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).
      ). More recently, a small-molecule screen was performed in the aim of identifying compounds binding HEPN with high affinity. This work showed preference of sacsin HEPN for compounds with a high number of phosphates (
      • Li X.
      • Ménade M.
      • Kozlov G.
      • Hu Z.
      • Dai Z.
      • McPherson P.S.
      • Brais B.
      • Gehring K.
      High-throughput screening for ligands of the HEPN domain of sacsin.
      ).
      Figure thumbnail gr1
      Figure 1Structure of the N-terminal Ubl domain of sacsin. A, domain architecture of sacsin consists of three SIRPT modules, N-terminal Ubl domain, and C-terminal DnaJ and HEPN domains. Structures of Ubl and the SIRPT1 Sr1 domain have been determined in this work. B, the Ubl domain crystallized as a swapped dimer partially stabilized by intramolecular Cys17–Cys20 disulfides. C, overlay of sacsin Ubl (green) and ubiquitin (magenta) shows similarity of the structures. D and E, comparison of β-sheet surfaces shows a more hydrophobic character of the ubiquitin site typically used for binding other proteins. The surface of the protein-binding site of ubiquitin shows more pronounced positive charge.
      Most of sacsin is composed of three large SIRPTs with each repeat divided into subrepeats, namely Sr1, Sr2, Sr3, and SrX (
      • Romano A.
      • Tessa A.
      • Barca A.
      • Fattori F.
      • de Leva M.F.
      • Terracciano A.
      • Storelli C.
      • Santorelli F.M.
      • Verri T.
      Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
      ). The second repeat does not contain SrX, making SIRPT2 smaller than SIRPT1 and SIRPT3. Within each of the SIRPTs, the first half of the Sr1 shows sequence homology to the nucleotide-binding domain (NBD) of Hsp90 (
      • Romano A.
      • Tessa A.
      • Barca A.
      • Fattori F.
      • de Leva M.F.
      • Terracciano A.
      • Storelli C.
      • Santorelli F.M.
      • Verri T.
      Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
      ).
      Hsp90 is a superfamily of chaperones that includes cytoplasmic Hsp90α and Hsp90β, glucose-regulated protein 94 (GRP94) localized in the endoplasmic reticulum, and mitochondrial tumor necrosis factor receptor–associated protein 1 (TRAP1) (
      • Seo Y.H.
      Organelle-specific Hsp90 inhibitors.
      ,
      • Shrestha L.
      • Patel H.J.
      • Chiosis G.
      Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease.
      ). Hsp90s comprise three functional domains starting with the NBD followed by a substrate-recognition domain and a dimerization domain at the C terminus (
      • Shrestha L.
      • Patel H.J.
      • Chiosis G.
      Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease.
      ). The NBDs adopt a Bergerat fold with a sandwich of four β-sheets and two α-helices forming a nucleotide-binding pocket (
      • Bergerat A.
      • de Massy B.
      • Gadelle D.
      • Varoutas P.C.
      • Nicolas A.
      • Forterre P.
      An atypical topoisomerase II from Archaea with implications for meiotic recombination.
      ). ATP binding and hydrolysis are required for Hsp90 chaperone function (
      • Seo Y.H.
      Organelle-specific Hsp90 inhibitors.
      ).
      The amino acid sequence of sacsin supports the hypothesis of a quality control function, but more studies are needed. Sr1 is a site for a number of disease-causing mutations: D168Y (
      • Vermeer S.
      • Meijer R.P.
      • Pijl B.J.
      • Timmermans J.
      • Cruysberg J.R.
      • Bos M.M.
      • Schelhaas H.J.
      • van de Warrenburg B.P.
      • Knoers N.V.
      • Scheffer H.
      • Kremer B.
      ARSACS in the Dutch population: a frequent cause of early-onset cerebellar ataxia.
      ), T201K (
      • Baets J.
      • Deconinck T.
      • Smets K.
      • Goossens D.
      • Van den Bergh P.
      • Dahan K.
      • Schmedding E.
      • Santens P.
      • Rasic V.M.
      • Van Damme P.
      • Robberecht W.
      • De Meirleir L.
      • Michielsens B.
      • Del-Favero J.
      • Jordanova A.
      • et al.
      Mutations in SACS cause atypical and late-onset forms of ARSACS.
      ), R272C (
      • Guernsey D.L.
      • Dubé M.P.
      • Jiang H.
      • Asselin G.
      • Blowers S.
      • Evans S.
      • Ferguson M.
      • Macgillivray C.
      • Matsuoka M.
      • Nightingale M.
      • Rideout A.
      • Delatycki M.
      • Orr A.
      • Ludman M.
      • Dooley J.
      • et al.
      Novel mutations in the sacsin gene in ataxia patients from Maritime Canada.
      ), R272H (
      • Romano A.
      • Tessa A.
      • Barca A.
      • Fattori F.
      • de Leva M.F.
      • Terracciano A.
      • Storelli C.
      • Santorelli F.M.
      • Verri T.
      Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
      ), and L308F (
      • Takado Y.
      • Hara K.
      • Shimohata T.
      • Tokiguchi S.
      • Onodera O.
      • Nishizawa M.
      New mutation in the non-gigantic exon of SACS in Japanese siblings.
      ). How these mutations contribute to ARSACS is unclear in the absence of structural information.
      Here, we present atomic structures of two domains of human sacsin: the N-terminal Ubl domain and the Sr1 domain of SIRPT1. The Ubl crystals contain a swapped dimer arrangement of the protein domain, which is potentially relevant in the context of the full-length protein. The Sr1 displays the Bergerat protein fold with a characteristic nucleotide-binding pocket but binds nucleotides only weakly. Importantly, the structure demonstrates that the pathological missense mutations, R272H, R272C, and T201K, affect the protein fold and/or stability and most probably target sacsin for proteasomal degradation. The ARSACS D168Y mutation likely affects chaperone activity via interference with ATP binding.

      Results

       Structure of the Ubl domain of sacsin

      To investigate sacsin function, we adopted a structure-directed approach. To elucidate the Ubl structure, we expressed sacsin (residues 2–85) in Escherichia coli, crystallized the purified protein, and obtained excellent X-ray diffraction. However, the structure could not be solved by molecular replacement with known ubiquitin or Ubl structures because sacsin Ubl has less than 30% sequence identity with ubiquitin. We sought to incorporate selenomethionine in the protein for experimental phasing, but the domain contains no methionines. We therefore made a series of mutants, based on sequence comparisons with other sacsin mammalian orthologs, to incorporate methionine: V9M, L26M, W29M, V34M, F39M, V46M, L58M, and L78M as well as a construct (residues 1–85) that includes the initiator methionine (M1). Of those mutants, only M1, L26M, F39M, and L78M were well expressed, and only M1 and L78M crystallized. Although the former did not yield any measurable anomalous scattering, the L78M mutant gave strong anomalous scattering and enabled the structure to be solved by multiwavelength anomalous dispersion. This mutant structure was subsequently used to determine the structure of native Ubl (Table 1).
      Table 1Data collection and refinement statistics
      UblUbl (L78M)Ubl (L78M)Sr1Sr1 (3-Met)Sr1 (4-Met)Anole Sr1
      Data collection
       Wavelength (Å)1.5420.9787 (peak)0.9792 (inflection)0.9770.9770.9770.977
       Space groupC2221C2221C2221P1P1P43C2
       Cell dimensions
      a, b, c (Å)51.9, 62.6, 90.852.1, 61.5, 91.652.1, 61.5, 91.641.1, 41.8, 105.941.7, 41.4, 74.170.8, 70.8, 100.9160.8, 38.2, 114.7
       α, β, γ (°)90, 90, 9090, 90, 9090, 90, 9097.9, 95.8, 107.299.6, 95.3, 107.590, 90, 9090, 131.6, 90
       Resolution (Å)50–2.10 (2.14–2.10)
      Highest-resolution shell is shown in parentheses.
      45.8–2.40 (2.53–2.40)36.6–2.40 (2.53–2.40)50–1.55 (1.58–1.55)50–1.90 (1.93–1.90)50–1.80 (1.83–1.80)50–1.85 (1.88–1.85)
      Rsym0.06 (0.29)0.061 (0.498)0.068 (0.707)0.05 (0.45)0.09 (0.46)0.10 (0.59)0.11 (0.57)
      II23.8 (7.3)24.8 (5.5)23.9 (4.3)34.6 (2.9)43.5 (4.5)47.0 (6.4)20.4 (2.9)
       Completeness (%)98.8 (82.1)98.9 (98.7)99.0 (98.6)96.8 (94.8)98.0 (96.9)100 (100)99.9 (100)
       Redundancy10.4 (8.1)14.0 (14.5)14.0 (14.5)4.3 (4.2)7.9 (7.5)15.2 (15.0)3.7 (3.7)
      Refinement
       Resolution (Å)26.0–2.1029.1–2.4050.0–1.5650.0–1.9150.0–1.8050.0–1.85
       No. reflections8,8435,92386,24732,56643,32343,261
      Rwork/Rfree0.187/0.2470.218/0.2840.182/0.2140.187/0.2260.198/0.2300.196/0.235
       No. atoms
       Protein1,2291,0635,6223,7093,9503,721
       Water5919390167203253
      B-factors
       Protein41.464.923.517.111.414.5
       Water45.257.539.237.325.633.5
       r.m.s. deviations
       Bond lengths (Å)0.0110.0090.0070.0110.0100.010
       Bond angles (°)1.271.11.161.371.341.28
       Ramachandran statistics (%)
       Most favored regions97.396.097.397.097.197.1
       Additional allowed regions2.74.02.73.02.92.9
      Deposition (PDB code)5VSX5VSZ5V445V455V465V47
      a Highest-resolution shell is shown in parentheses.
      The structure revealed the β-grasp fold characteristic of ubiquitin-like domains (Fig. 1B). Surprisingly, although the fold is identical to ubiquitin, the structure contains an unusual feature. The Ubl molecules form a dimer through swapping of their N-terminal β-strands. This dimeric arrangement is likely stabilized by an intramolecular disulfide bond between Cys17 and Cys20 (Fig. 1B). The dimerization caused extensive intermolecular contacts via β-sheet surfaces. This is unexpected because this surface typically contains a hydrophobic patch used for recognition of binding partners, including proteasome subunits. This hinted that the observed dimer could be a crystallization artifact; domain swapping often occurs unnaturally during crystallization of truncated proteins (
      • Liu Y.
      • Eisenberg D.
      3D domain swapping: as domains continue to swap.
      ).
      To experimentally determine the aggregation state of the Ubl domain in solution, we used multiangle light scattering coupled with size-exclusion chromatography to measure the molecular mass of 10.5 kDa, which is in a good agreement with the expected mass of 9.9 kDa for monomeric Ubl (Fig. S1). We repeated this experiment with a larger fragment of sacsin (residues 1–339) that includes the Ubl and the following Hsp90-like Sr1 domain. Again, the experimental molecular mass (35.5 kDa) correlated well with the calculated mass of the monomer (38.7 kDa). In conclusion, the isolated Ubl domain does not dimerize in solution, confirming that the swapped dimer observed in the Ubl crystal is likely a crystallization artifact.
      When reconstructed from the swapped dimer, the structure of the sacsin Ubl monomer is very similar to ubiquitin with a root mean square deviation (r.m.s.d.) of 1.7 Å over 61 Cα atoms (Fig. 1C). Nevertheless, the surface composition is significantly different. Ubiquitin utilizes a highly conserved surface centered around Ile44 to recognize ubiquitin-binding proteins. In ubiquitin, the side chains of Ile44, Leu8, and Val70 form a hydrophobic patch, whereas Arg42 and His68 are responsible for charged intermolecular contacts (Fig. 1, D and E). In sacsin, only Arg50 is conserved. Ile44 is substituted by Trp52, which is hydrophobic but has a bulkier side chain, possibly affecting selection of binding partners. Two other hydrophobic residues found in ubiquitin (Val70 and Leu8) are replaced by Gln79 and Gly16 in sacsin. These differences result in surface-charge differences: the surface of ubiquitin has a much more pronounced positive charge (Fig. 1, D and E).

       High-resolution structure of the Sr1 domain of sacsin SIRPT1 repeat

      Based on a bioinformatics analysis of sacsin (
      • Romano A.
      • Tessa A.
      • Barca A.
      • Fattori F.
      • de Leva M.F.
      • Terracciano A.
      • Storelli C.
      • Santorelli F.M.
      • Verri T.
      Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
      ), we designed several constructs of the sacsin Hsp90-like domain and screened them for crystallization. The first Sr1 domain (residues 89–336) (Fig. 2A) of the first SIRPT yielded well diffracting crystals. Because no protein structure with high enough sequence homology was available in the PDB, the sacsin Sr1 fragment was labeled with selenomethionine for experimental phasing of diffraction data. The crystallized construct naturally contains two methionine residues, Met148 and Met214, but the anomalous signal was not sufficient to solve the phase problem. Again, a series of mutants was generated where multiple residues were substituted with methionine. Among these, the F270M/K291M/L318M (3-Met) and I113M/F270M/K291M/L308M (4-Met) mutants yielded well diffracting crystals. The 3-Met mutant crystallized in the same space group as the native protein but with different dimensions, whereas the 4-Met mutant crystallized in P43. The structure of the mutants was solved by single-wavelength anomalous dispersion and subsequently used for phasing of native data at 1.56-Å resolution (Table 1). We also crystallized and determined the structure of the homologous (91% identity) domain of sacsin from the green anole. This protein crystallized in yet another crystal form (space group C2). The crystals of WT human Sr1 contain three protein molecules in the asymmetric unit, whereas the other crystal forms have two molecules in the asymmetric unit. Having multiple crystal forms with each containing two or three molecules in the asymmetric unit is helpful for identifying regions of flexibility and bias resulting from crystal contacts.
      Figure thumbnail gr2
      Figure 2Structure of sacsin Sr1 domain. A, domain architecture highlighting the SIRPT1 Sr1 domain. B, cartoon representation of human sacsin Sr1 domain (residues 89–336) colored from N terminus (blue) to C terminus (red). C, the N-terminal strands in the crystals are swapped. D, side chains of Phe89 and Gln91 are inserted into the surface of another Sr1 molecule.
      The Sr1 structures show an α/β-sandwich where a central β-sheet is covered by α-helical bundles on both sides (Fig. 2B). The eight-stranded β-sheet is largely antiparallel except for strands β1 and β7, which are parallel to each other. The overall structure of the Sr1 domain is similar to a Bergerat fold (
      • Bergerat A.
      • de Massy B.
      • Gadelle D.
      • Varoutas P.C.
      • Nicolas A.
      • Forterre P.
      An atypical topoisomerase II from Archaea with implications for meiotic recombination.
      ), which is characteristic of nucleotide-binding domains involved in ATPase activity, in particular Hsp90 family proteins. A structure similarity search using the DALI database returned Hsp90 orthologs in the endoplasmic reticulum (GRP94), mitochondria (TRAP1), and cytoplasm (Hsp90) (Fig. S2). Comparison with these structures shows that similarity is mostly limited to the central β-sheet and the α-helices forming the nucleotide-binding site. Interestingly, the opposite side of the β-sheet is mostly solvent-accessible in the homologous structures, whereas it is covered by a four-helical bundle in sacsin.
      Comparison of the multiple Sr1 molecules shows that the independently folded region extends from Pro94 to Ser335. Nevertheless, the majority of molecules (seven of nine) have the N terminus structured from Phe89, and in some, even the preceding linker is visible in electron density maps. This is because the N-terminal part is involved in crystal contacts largely driven by Phe89 inserting into a hydrophobic pocket proximal to the putative nucleotide-binding site (Fig. 2, C and D). This is somewhat reminiscent of the TRAP1 structure where the N-terminal parts of nucleotide-binding domains were swapping and straddling the neighboring subunits of TRAP1 dimer (
      • Sung N.
      • Lee J.
      • Kim J.H.
      • Chang C.
      • Tsai F.T.
      • Lee S.
      2.4 Å resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix.
      ). It is difficult to evaluate the relevance of this observation in the context of the isolated Sr1 domain. The Sr1 domain does not dimerize in solution, but full-length sacsin is likely a dimer because the C-terminal HEPN domain must dimerize to fold properly (
      • Kozlov G.
      • Denisov A.Y.
      • Girard M.
      • Dicaire M.J.
      • Hamlin J.
      • McPherson P.S.
      • Brais B.
      • Gehring K.
      Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).
      ). Therefore, it is possible that these intermolecular contacts are relevant in the context of full-length sacsin. Another possibility is that the identified hydrophobic pocket has a chaperone function by binding exposed hydrophobic residues of protein substrates.
      All the SIRPT1 Sr1 structures are highly similar. The r.m.s.d. between the human Sr1 structures is 0.3–0.4 Å and between human and green anole Sr1 is ∼0.6 Å (Fig. S3). The main differences between the Sr1 structures are in the loop corresponding to the ATP lid of Hsp90. This loop (residues 174–191) is completely disordered in some Sr1 molecules, partially disordered in two molecules, and well ordered in the P43 crystal form, which is missing only Asp182 at the very tip of the loop. This is a result of the crystal packing where conformation of this loop is stabilized via intermolecular crystal contacts. It is worth noting that, in the P43 crystals, this loop appears as a β-hairpin. This is in contrast to the helix-loop-helix arrangement observed in Hsp90 structures (Fig. S2). This helix-loop-helix is a flexible element from Hsp90, which closes over the bound ATP; it exposes hydrophobic residues to allow the dimerization crucial for the enzymatic activity of the protein (
      • Ali M.M.
      • Roe S.M.
      • Vaughan C.K.
      • Meyer P.
      • Panaretou B.
      • Piper P.W.
      • Prodromou C.
      • Pearl L.H.
      Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex.
      ,
      • Prodromou C.
      The ‘active life’ of Hsp90 complexes.
      ).
      Given the sequence similarity of the solved fragment with Sr1 modules from SIRPT2 and SIRPT3 (∼35% sequence identity), the structures of these modules are expected to be very similar (Fig. S4). Thus, our SIRPT1 Sr1 structures are good templates for interpreting structure–function studies of the other Sr1 modules.

       Sacsin Sr1 binds nucleotides with low affinity

      Because structurally related Hsp90-like domains bind ATP and possess ATPase activity, we sought to test nucleotide binding to SIRPT1 Sr1. We chose NMR because it is sensitive to even low-affinity binding, and Sr1 gives excellent NMR spectra. The 1H-15N correlation spectrum of Sr1 shows good dispersion of signals characteristic of a well folded protein. We first tested Sr1 for binding to ADP in the presence of Mg2+ as Hsp90 displays 4-fold higher affinity to ADP than to ATP (29 versus 132 μm) (
      • Prodromou C.
      • Roe S.M.
      • O'Brien R.
      • Ladbury J.E.
      • Piper P.W.
      • Pearl L.H.
      Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.
      ). If bound, ADP would cause shifts for a number of signals in the nucleotide-binding site. Somewhat unexpectedly, only high concentrations of ADP resulted in spectral changes, suggesting a Kd of over 10 mm (Fig. 3A). Similar results were obtained in a titration with ATP (data not shown). This is consistent with no binding observed in isothermal titration calorimetry (ITC) experiments (Fig. S5). We sought to explain this marginal affinity for nucleotides by examining the nucleotide-bound structures of Hsp90-like domains.
      Figure thumbnail gr3
      Figure 3Sacsin Sr1 binds nucleotides with low affinity. A, overlays of the 1H-15N correlation NMR spectra show shifts of some peaks upon addition of ADP at high concentrations. B and C, comparison of the Sr1 structure with nucleotide-bound TRAP1 shows conservation of many residues involved in nucleotide binding and hydrolysis. D, a steady-state ATPase activity assay shows low activity for the sacsin Sr1 domain alone and as a GST fusion. GST was used as a negative control, and Hsc70 (including sacsin DnaJ and C-Bag1) was used as a positive control.
      The Bergerat fold is characterized by several conserved motifs involved in binding and hydrolysis of ATP (
      • Bergerat A.
      • de Massy B.
      • Gadelle D.
      • Varoutas P.C.
      • Nicolas A.
      • Forterre P.
      An atypical topoisomerase II from Archaea with implications for meiotic recombination.
      ). Structural comparison of Sr1 with nucleotide-bound TRAP1 shows that some features are conserved in sacsin (Fig. 3, B and C). Namely, motif I includes Glu116 that is expected to serve as a nucleophile, whereas Asn120 could help coordinate the Mg2+ ion involved in ATP binding. Thr269 in motif IV could be potentially engaged in a polar contact with the N1 atom of the adenine ring. The other differences may negatively affect nucleotide binding. In motif II, the typical aspartate residue is replaced by Asn160 in sacsin. The role of this residue is to form a hydrogen bond with the N6 amino group of adenine, and asparagine will not fully replace aspartate for this function. Another important substitution is Phe164 in place of a conserved glycine. The presence of glycine is thought to provide space for the adenine ring; the bulky phenylalanine side chain would not do this, although it is possible that it could provide aromatic stacking with adenine. Finally, motif IV is part of a loop in Hsp90-like proteins that acts as a lid on the nucleotide-binding site and clamps the γ-phosphate of ATP. In TRAP1, the loop comprises two α-helices and has conserved glycines on the interface with bound nucleotide. The corresponding loop in sacsin is shorter and does not possess corresponding glycine residues. In the P43 crystal form of sacsin Sr1, this loop adopts a defined but irregular conformation, suggesting this stretch does not have propensity to form α-helices. Although the loop in Sr1 appears unlikely to support nucleotide binding, it is possible that another part of sacsin could function as an ATP-binding lid. All this points to insufficiency of Sr1 for ATPase activity. In agreement, steady-state ATPase assays did not show appreciable activity for the sacsin Sr1 domain (Fig. 3D).
      If the putative nucleotide-binding pocket in Sr1 were functionally relevant, one would expect the above mentioned residues to be conserved in the other SIRPTs. Indeed, Glu116, Asn120, Asn160, Thr269, and even the somewhat puzzling Phe164 are all invariantly conserved in all three SIRPTs (Fig. S4). The SIRPT1 Sr1 structure suggests that none of the residues play a structural role in the protein; therefore, their conservation in Sr1 domains is likely functional for ATP binding and hydrolysis by full-length sacsin.

       Role of pathological mutations

      Five ARSACS missense mutations (D168Y, T201K, R272H, R272C, and L308F) occur in SIRPT1 Sr1. We generated these mutants in the context of the isolated Sr1 domain (Fig. 4A). The D168Y and L308F mutations produced soluble protein in yields similar to the WT construct. Expression levels of the other three mutants were severely reduced (Fig. 4E). We used thermal shift assays (also called differential scanning fluorimetry) to measure the thermal stability of the D168Y and L308F mutants and compared them with the WT protein. The conservative L308F mutation slightly lowered the melting temperature compared with the WT protein (51 versus 52 °C), whereas the D168Y mutation (46 °C) significantly destabilized the protein (Fig. S6).
      Figure thumbnail gr4
      Figure 4ARSACS-causing mutations in Sr1. A, positions of pathological mutations in the Sr1 structure. B, side chain of Thr201 is buried while forming hydrogen bonds with backbone of Val203 and Pro274. C, Asp168 is positioned in the nucleotide-binding site, and its mutation to tyrosine likely prevents nucleotide binding. The superposed structure of TRAP1 (PDB code 5F5R; light purple) with an ATP analog and Mg2+ bound is shown. D, side chain of Arg272 is buried while forming hydrogen bonds with side chain of Tyr157, carbonyls of Tyr151 and Gly153, and two water molecules. E, SDS-PAGE analysis shows lack of protein expression of R272C and R272H Sr1 domain mutants in multiple clones.
      These results correlate well with the structure. Thr201 is buried, and introducing a lysine side chain would disrupt the hydrophobic core of the protein (Fig. 4B). Arg272 is also buried; its guanidinium group makes hydrogen bonds to carbonyls of Tyr151 and Gly153 and to the side chain of Tyr157 (Fig. 4D). Mutating Arg272 to histidine or cysteine abrogates these contacts and would be expected to destabilize the protein. Asp168 and Leu308 are more intriguing as both residues are solvent-exposed, and their disease-related mutations do not cause structural changes. These residues could be involved in functional aspects of sacsin. In particular, Asp168 is positioned in the putative nucleotide-binding site, and introduction of bulky tyrosine side chain could severely interfere with nucleotide binding (Fig. 4C). Thr201, Arg272, and Asp168 are invariantly conserved in all SIRPTs, highlighting their structural and functional importance (Fig. S4).
      The function of Leu308 is less clear; it is located on a small hydrophobic patch on the domain surface and may be involved in binding substrates or another domain of sacsin. Leu308 is substituted with isoleucine in SIRPT2 and methionine in SIRPT3, which leaves unanswered the question of why the L308F mutation causes ARSACS.

       Conformation of Ubl-Sr1 in solution

      Having solved the Ubl and Sr1 structures separately, we wondered whether they orient independently in solution or form a relatively rigid tandem. To address this, we performed small-angle X-ray scattering (SAXS) experiments with the sacsin fragment containing both domains (residues 1–339). SAXS can be used to calculate the radius of gyration (Rg) and generate molecular shape reconstructions and analyze dynamics (
      • Putnam C.D.
      • Hammel M.
      • Hura G.L.
      • Tainer J.A.
      X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.
      ,
      • Bernadó P.
      • Mylonas E.
      • Petoukhov M.V.
      • Blackledge M.
      • Svergun D.I.
      Structural characterization of flexible proteins using small-angle X-ray scattering.
      ). We collected scattering data on Ubl-Sr1 at concentrations up to 6 mg/ml (Fig. S7). The Ubl-Sr1 fragment showed good behavior with no apparent aggregation as attested by the linearity of the Guinier plot. The estimated molecular mass from the SAXS data is 44.5 kDa, which is consistent with the theoretical mass of 38.7 kDa. This is in agreement with multiangle light scattering data for the same construct.
      We took advantage of the structure of individual domains to estimate the degree of flexibility using dynamic ensemble optimization (
      • Tria G.
      • Mertens H.D.
      • Kachala M.
      • Svergun D.I.
      Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering.
      ). Briefly, a pool of 10,000 structures for the Ubl and Sr1 domains linked by a flexible tether was generated, and subsets from this pool were selected to fit the SAXS data. The distributions of Rg from the fitted ensembles in two independent runs are narrower than that of the random pool (Fig. S7), suggesting that the two domains adopt a compact configuration. The statistical factor Rflex of 67% is less than that for the random distribution (88%), and Rσ is slightly less than 1, suggesting that the system has limited flexibility. Starting from this observation, we used rigid-body modeling with CORAL to determine the global shape of the fragment (
      • Petoukhov M.V.
      • Franke D.
      • Shkumatov A.V.
      • Tria G.
      • Kikhney A.G.
      • Gajda M.
      • Gorba C.
      • Mertens H.D.
      • Konarev P.V.
      • Svergun D.I.
      New developments in the ATSAS program package for small-angle scattering data analysis.
      ). The structures of Ubl and Sr1 were used to define the boundaries of the rigid domains and length of the interdomain linker. Superposition of the 30 lowest-energy models using the Sr1 model revealed that 90% of them converged to one cluster (Figs. 5 and S7). Altogether, the cluster showed a good fit between the predicted and observed scattering (Fig. 5). Although the calculations converged to a single orientation, we do not believe that the Ubl-Sr1 domains necessarily form a rigid pair in solution as NMR titrations of 15N-labeled Ubl domain with Sr1 did not detect an interaction (data not shown). However, transient interactions between Ubl and Sr1 are very likely considering the short length of the linker between the domains.
      Figure thumbnail gr5
      Figure 5SAXS analysis of sacsin Ubl-Sr1 domains. A, SAXS data and best rigid-body fit with the Ubl and Sr1 domains using CORAL. B, the best rigid-body model from CORAL (cartoon) was fitted into pseudodensity derived from ab initio modeling (white surface) contoured at the Porod volume.

      Discussion

      In the 20 years since the discovery of sacsin, progress in its structural characterization has been relatively limited. The full-length protein is very challenging to study due to its large size and low level of expression. At the same time, the success of the divide-and-conquer approach typically used by structural biologists has been restricted to only a few regions. The reasons include difficulties in defining the boundaries of the independently folded domains as well as the possible requirement for interactions between distant parts of sacsin for protein expression or stability. Before this study, the structures of only two domains have been determined: the HEPN domain (
      • Kozlov G.
      • Denisov A.Y.
      • Girard M.
      • Dicaire M.J.
      • Hamlin J.
      • McPherson P.S.
      • Brais B.
      • Gehring K.
      Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).
      ) and DnaJ (PDB code 1IUR). Here, we double the number of known structures. Although the SIRPT1 Sr1 structure accounts for only 5.4% of amino acids in sacsin, the expected structural similarity to the Sr1 domain in SIRPT2 and SIRPT3 increases the coverage to ∼16%. With the N-terminal Ubl domain and previous structures, we now have structural knowledge of close to 25% of this gigantic protein.
      Sacsin is expected to be a dimer in solution due to dimerization of its C-terminal HEPN domain (
      • Kozlov G.
      • Denisov A.Y.
      • Girard M.
      • Dicaire M.J.
      • Hamlin J.
      • McPherson P.S.
      • Brais B.
      • Gehring K.
      Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).
      ). There are still numerous uncharacterized regions in sacsin that could also dimerize. Although the isolated sacsin Ubl does not form a stable dimer in solution, it could form the swapped dimer observed in the protein crystals in the context of an already dimeric protein. Hsp90s are dimers, so it would not be surprising if the Sr1 domains were associated with as-yet-uncharacterized dimerization domains in sacsin.
      The initial suggestion that sacsin is a putative chaperone came from identification of DnaJ and Hsp90-like sequences (
      • Engert J.C.
      • Bérubé P.
      • Mercier J.
      • Doré C.
      • Lepage P.
      • Ge B.
      • Bouchard J.P.
      • Mathieu J.
      • Melançon S.B.
      • Schalling M.
      • Lander E.S.
      • Morgan K.
      • Hudson T.J.
      • Richter A.
      ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF.
      ). This was followed by the demonstration of activity of the sacsin DnaJ domain in E. coli cells and ATPase and chaperone activity of the SIRPT1 fragment (
      • Anderson J.F.
      • Siller E.
      • Barral J.M.
      The neurodegenerative-disease-related protein sacsin is a molecular chaperone.
      ,
      • Anderson J.F.
      • Siller E.
      • Barral J.M.
      The sacsin repeating region (SRR): a novel Hsp90-related supra-domain associated with neurodegeneration.
      ). Although our work shows that an isolated Sr1 domain does not bind nucleotides with appreciable affinity, this is not inconsistent with Sr1 ATPase activity in the context of the larger SIRPT fragment or full-length sacsin. Similar uncertainties arose before the definitive demonstration of ATP binding by the Hsp90 NBD domain (
      • Prodromou C.
      • Roe S.M.
      • O'Brien R.
      • Ladbury J.E.
      • Piper P.W.
      • Pearl L.H.
      Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.
      ). Several facts argue for Sr1 ATPase activity. First, similarity between Sr1 and Hsp90 proteins is highest in the nucleotide-binding site of the NBD. Many of the residues involved in ATP binding and hydrolysis in Hsp90 proteins are conserved in all three Sr1 domains (Fig. S4). Sr1 domains have the highest occurrence of ARSACS-causing missense mutations among all the known sacsin domains, which underlines their functional importance (
      • Romano A.
      • Tessa A.
      • Barca A.
      • Fattori F.
      • de Leva M.F.
      • Terracciano A.
      • Storelli C.
      • Santorelli F.M.
      • Verri T.
      Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
      ). Second, the D168Y mutation decreased ATP hydrolysis by SIRPT1 (residues 1–1456) (
      • Anderson J.F.
      • Siller E.
      • Barral J.M.
      The sacsin repeating region (SRR): a novel Hsp90-related supra-domain associated with neurodegeneration.
      ), which is consistent with a role in catalysis.
      Given that SIRPT modules make up more than 80% of this large protein, it is likely that the SACS gene was formed via triplication of the SIRPT region. The Sr1 domains appear not to be functionally redundant considering that single mutations in any Sr1 lead to disease. The activity of all three modules is necessary to perform the protein function. There are six Sr1 modules in a sacsin dimer, possibly arranged as three pairs. To our knowledge, there have been no reports of dominant-negative alleles among ARSACS patients.
      The importance of Ubl domain is less clear as no pathological missense mutations have been found in that domain. The HEPN domain would appear to be essential based on the pathology of a missense mutation (N4549D), but it is possible that the mutation has indirect effects on the protein expression or stability (
      • Richter A.M.
      • Ozgul R.K.
      • Poisson V.C.
      • Topaloglu H.
      Private SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) families from Turkey.
      ).
      What is the function of yet uncharacterized sacsin domains? By analogy with Hsp90 proteins, the Sr2 modules, which directly follow Sr1s, could serve as substrate-binding domains. It is clear that more studies are needed to assign functions to the many parts of sacsin for which we have no structural information. Ultimately, we need to understand how the global structure of sacsin defines and affects its function. The structural characterization of larger multidomain fragments will be particularly important for understanding sacsin and identifying avenues for the treatment of ARSACS.

      Experimental procedures

       Cloning, protein expression, and purification

      The Ubl (residues 2–85), Sr1 (residues 89–336), and Ubl-Sr1 (residues 1–339) domains were cloned by PCR using human cDNA as a template in pGEX-6P-1 (Amersham Biosciences) between BamHI and XhoI. The site-directed mutants of the Ubl and Sr1 domains were via standard site-directed mutagenesis using mismatched primers and PCR with TaqPlus Precision polymerase (Stratagene, La Jolla, CA) following the manufacturer's protocol. Automated DNA sequencing was performed to verify all sequence modifications.
      Proteins were expressed in a BL21 E. coli strain. The cells were grown at 37 °C in Luria broth to an optical density of 0.8, and expression was induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside at 30 °C for 4 h or 18 °C overnight. After centrifuging the cells, the pellets were resuspended in phosphate-buffered saline (PBS) (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4, pH 7.4) containing 1 mm phenylmethylsulfonyl fluoride, 0.1 mg/ml lysozyme, and 80 units of DNase and lysed by sonication. Cell debris was removed by centrifugation, and the GSH S-transferase (GST) fusion protein was purified using GSH-agarose resin (Pierce). After eluting the proteins in PBS containing 20 mm GSH, the tag was removed by cleavage with the 3C rhinovirus protease. The proteins were additionally purified by size-exclusion chromatography on a Superdex75 gel filtration column (GE Healthcare) in Ubl buffer (10 mm HEPES, pH 7.0, 50 mm NaCl, 1 mm DTT) or Sr1 buffer (20 mm HEPES, pH 7.0, 100 mm NaCl, 3 mm DTT).
      For 15N labeling, the cells were grown in M9 minimal medium supplemented with [15N]ammonium chloride as the sole source of nitrogen. For selenomethionine labeling, the plasmid was transformed into a methionine auxotroph DL41 (DE3) E. coli strain, and the cells were grown in LeMaster medium supplemented with selenomethionine. The expression and purification protocols were the same as for the native protein.

       Crystallization, data collection, and processing

      The Ubl domain (WT or SeMet-L78M) was crystallized using the hanging drop vapor diffusion method by equilibrating a 1.5-μl drop of protein (3.2 mg/ml) with 1.5 μl of reservoir solution containing 250 mm NaCl, 100 mm Bicine, pH 8.6, 1 mm lead acetate, and 10% glycerol. Ubl crystals grew within 16 h at 22 °C. For data collection, glycerol was added to a final concentration of 25% for cryoprotection.
      For the Sr1 domain, initial conditions were identified by sitting drop vapor diffusion using the Classics II screen (Qiagen). The best crystals were obtained by equilibrating a 1.0-μl drop of protein (17 mg/ml) in 20 mm HEPES, pH 7.0, 100 mm NaCl, and 3 mm DTT with 1.0 μl of reservoir solution containing 0.1 m MES, pH 6.5, 28% (w/v) PEG 3350, and 0.2 m ammonium acetate with the drop suspended over 1 ml of reservoir solution. Selenomethionine-labeled proteins were crystallized in similar conditions. Sr1 crystals grew in 3–6 days at 22 °C. For data collection, 20% glycerol was added for cryoprotection, and the crystals were flash cooled in an N2 cold stream.
      The crystals were cryoprotected with 10–25% glycerol or ethylene glycol and flash-cooled in an N2 cold stream. X-ray diffraction data were collected using an ADSC Quantum 210 CCD detector (Area Detector Systems Corp.) on beamline A1 at the Cornell High-Energy Synchrotron Source (CHESS) at 0.9770 Å. Data processing and scaling were performed with HKL2000 (Table 1) (
      • Otwinowski Z.
      • Minor W.
      Processing of X-ray diffraction data collected in oscillation mode.
      ).

       Structure determination and refinement

      Diffraction data from the selenomethionine-labeled crystals of Ubl L78M (residues 2–85) were collected using three wavelengths at the selenium adsorption edge, inflection, and remote positions on an ADSC Quantum-210 CCD detector (Area Detector Systems Corp.) at beamline F2 at CHESS. The SeMet-labeled crystals diffracted up to 2.4 Å at synchrotron. Despite the presence of lead acetate in the crystallization conditions as an additive, lead ions were not incorporated in the crystal lattice and did not yield anomalous signal. Instead, the structure was determined using selenium multiangle dispersion. The data were phased with PHENIX using Autosol and the peak/inflection data sets (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). The Ubl L78M structure served as the model for molecular replacement for the previously collected 2.1-Å data on the native WT Ubl (residues 2–85) crystals. The partial model thus obtained was extended manually with the program Coot and improved by refinement using REFMAC and model refitting followed by TLS (translation-libration-screw) refinement (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      Refinement of macromolecular structures by the maximum-likelihood method.
      ,
      • Winn M.D.
      • Murshudov G.N.
      • Papiz M.Z.
      Macromolecular TLS refinement in REFMAC at moderate resolutions.
      ,
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ).
      Diffraction data from the Sr1 (residues 89–336) crystals were collected using a single-wavelength (0.977 Å) anomalous dispersion regime on an ADSC Quantum-210 CCD detector at the A1 beamline at CHESS (Table 1). Data processing and scaling were performed with HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of X-ray diffraction data collected in oscillation mode.
      ). Diffraction data of the 3-Met and 4-Met Sr1 mutants were phased using anomalous signal from selenium atoms by the single-wavelength anomalous dispersion method with the program PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). The initial model was built using PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ), improved using Coot (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ), and further refined using Refmac5 (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      Refinement of macromolecular structures by the maximum-likelihood method.
      ). Data from the WT human and lizard Sr1 constructs were phased by molecular replacement using the 3-Met Sr1 structure as the search model. The initial model was adjusted and completed with Coot (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ). The final models were improved by several cycles of refinement using Refmac5 (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      Refinement of macromolecular structures by the maximum-likelihood method.
      ). The refinement statistics are shown in Table 1.
      The final models have no outliers in the Ramachandran plot computed using PROCHECK. Coordinates of the WT Ubl domain, L78M Ubl mutant, WT Sr1, 3-Met and 4-Met Sr1 mutants, and the anole Sr1 domain have been deposited in the Protein Data Bank with the accession codes 5VSX, 5VSZ, 5V44, 5V45, 5V46, and 5V47, respectively.

       Multiangle light scattering measurements

      Size-exclusion chromatography and multiangle light scattering experiments were performed using an analytical Superose 6 column (GE Healthcare) equilibrated in 20 mm HEPES, pH 7.0, 100 mm NaCl, and 3 mm DTT with in-line static light scattering by absorbance and differential refractive index detectors (miniDAWN TREOS and Optilab-rEX, Wyatt Technology Corp.). The sacsin Ubl-Sr1 was loaded at 7 mg/ml and Ubl was loaded at 7.4 mg/ml with a sample size of 50 μl at 22 °C. Data were analyzed with the ASTRA software package (Wyatt Technology Corp.).

       NMR spectroscopy

      NMR titration experiments were performed at 298 K on a Bruker 600-MHz spectrometer. For titrations with nucleotides, NMR samples of the 15N-labeled Sr1 (residues 89–336) construct were prepared as 0.18–0.20 mm in 90% buffer A and 10% D2O. 15N-1H heteronuclear single quantum correlation spectroscopy (HSQC) titrations were performed by stepwise addition of ATP or ADP to a final nucleotide concentration of 100 mm. The NMR spectra were processed by NMRPipe (
      • Delaglio F.
      • Grzesiek S.
      • Vuister G.W.
      • Zhu G.
      • Pfeifer J.
      • Bax A.
      NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
      ) and analyzed using SPARKY (
      • Lee W.
      • Tonelli M.
      • Markley J.L.
      NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy.
      ).

       Isothermal titration calorimetry

      ITC experiments were performed on a MicroCal VP-ITC titration calorimeter (Malvern Instruments Ltd.). The sample cell was loaded with 30 μm Sr1 domain, and the syringe contained 0.3 mm nucleotide. All experiments were carried out at 293 K with 19 injections of 15 μl with stirring at 310 rpm. Results were analyzed using Origin software (MicroCal).

       Steady-state ATPase assays

      Steady-state ATPase experiments were based on published procedures (
      • Tzankov S.
      • Wong M.J.
      • Shi K.
      • Nassif C.
      • Young J.C.
      Functional divergence between co-chaperones of Hsc70.
      ). Reactions contained 4 μm Hsc70, 4 μm GST-sacsin DnaJ, and 4 μm C-terminal domain of Bag1 or 8 μm either GST-Sr1, Sr1, or GST supplemented with 2 mm ATP and 5 μCi/ml [α-32P]ATP (PerkinElmer Life Sciences). Reactions were assembled on ice and initiated by incubation at 30 °C. Aliquots were taken at various time points, inactivated with 40 mm EDTA, then loaded on polyethyleneimine cellulose TLC plates (Fisher), and developed in 0.5 m LiCl and 0.5 m formic acid. The plates were exposed on a phosphorimaging screen, and the ADP produced was quantified with a Typhoon scanner and ImageQuant software (GE Healthcare). Linear ATPase rates were calculated by regression as mol of ADP/(mol of enzyme × min).

       Thermal shift assays

      Each reaction contained a total 20 μl of solution with 20 μm WT or mutant Sr1 (residues 89–336) in buffer and 1× Protein Thermal Shift dye (Protein Thermal Shift Dye kit, Life Technologies). Samples were heated from 25 to 99 °C at a rate of 1 °C/min, and fluorescence signals were monitored by the StepOne Plus quantitative real-time PCR system (Life Technologies). All measurements were done in triplicates. Data were analyzed using Thermal Shift software (Life Technologies). The maximum change of fluorescence with respect to temperature was used to determine the melting temperature (Tm).

       SAXS

      Small-angle X-ray scattering data were collected on SIBYLS beamline (12.3.1) at the Advanced Light Source at Lawrence Berkeley National Laboratory. Scattering data were collected for 0.5–1 s at 20 °C at protein concentrations of 1, 2, and 3 mg/ml for the Ubl-Sr1 construct. Background scattering from the buffer (20 mm HEPES, pH 7.0, 100 mm NaCl, 2% glycerol, 3 mm DTT) was measured for 0.5–1 s. Scaling, buffer subtraction, and merging were performed using Primus, part of the ATSAS package (
      • Petoukhov M.V.
      • Franke D.
      • Shkumatov A.V.
      • Tria G.
      • Kikhney A.G.
      • Gajda M.
      • Gorba C.
      • Mertens H.D.
      • Konarev P.V.
      • Svergun D.I.
      New developments in the ATSAS program package for small-angle scattering data analysis.
      ). The pair-distance distribution was calculated using GNOM (
      • Svergun D.I.
      Determination of the regularization parameter in indirect-transform methods using perceptual criteria.
      ). Molecular mass was estimated using the Porod volume method (
      • Petoukhov M.V.
      • Franke D.
      • Shkumatov A.V.
      • Tria G.
      • Kikhney A.G.
      • Gajda M.
      • Gorba C.
      • Mertens H.D.
      • Konarev P.V.
      • Svergun D.I.
      New developments in the ATSAS program package for small-angle scattering data analysis.
      ).
      For rigid-body modeling and dynamics ensemble analysis, the Ubl and Sr1 domains had to be modified. Briefly, a monomeric model of the sacsin Ubl (residues 6–79) was built by merging the first β-strand from chain A (residues 6–14) and the remainder of the structure from chain B (residues 20–79). The loop (residues 15–19) was built and regularized using Coot. The Sr1 was modified by building a loop between residues 174 and 190 for which no electron density was visible. The two domains were connected by a 14-residue flexible linker. EOM 2.0 was used to generate 10,000 structures using the two domains using the genetic algorithm for conformer selection. The genetic algorithm was performed 100 times twice to estimate the variability in the distribution of Rg values. The Rflex of 67% is less than that for the random distribution (88%), and Rσ is slightly less than 1, suggesting that the system has limited flexibility (
      • Bernadó P.
      • Mylonas E.
      • Petoukhov M.V.
      • Blackledge M.
      • Svergun D.I.
      Structural characterization of flexible proteins using small-angle X-ray scattering.
      ). Rigid-body modeling of the SAXS data was performed using the software CORAL (
      • Petoukhov M.V.
      • Franke D.
      • Shkumatov A.V.
      • Tria G.
      • Kikhney A.G.
      • Gajda M.
      • Gorba C.
      • Mertens H.D.
      • Konarev P.V.
      • Svergun D.I.
      New developments in the ATSAS program package for small-angle scattering data analysis.
      ) using the same domain as described above. Thirty models with χ2 < 2.0 were generated. Ab initio modeling was performed using DAMMIF (
      • Franke D.
      • Svergun D.I.
      DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering.
      ). Twenty models were generated and averaged using DAMAVER. The resulting coordinates were used to generate pseudodensities using Situs-pdb2vol (
      • Wriggers W.
      • Chacón P.
      Modeling tricks and fitting techniques for multiresolution structures.
      ) and contoured at the particle volume derived from the Porod invariant using UCSF-Chimera (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera—a visualization system for exploratory research and analysis.
      ).

      Author contributions

      M. M., G. K., J.-F. T., and K. G. conceptualization; M. M., G. K., J.-F. T., H. P., S. S., S. W., X. L., H. H., and M. J. H. W. data curation; M. M., G. K., J.-F. T., H. P., S. S., S. W., X. L., and M. J. H. W. formal analysis; M. M., G. K., J.-F. T., H. P., S. S., S. W., X. L., H. H., M.-J. D., and M. J. H. W. investigation; M. M. and G. K. writing-original draft; M. M. and G. K. project administration; G. K., J.-F. T., B. B., P. S. M., J. C. Y., and K. G. writing-review and editing; M.-J. D. resources; B. B., P. S. M., J. C. Y., and K. G. funding acquisition; K. G. supervision.

      Acknowledgments

      We thank Tin Pan for technical assistance. Data were acquired at the Macromolecular Diffraction (MacCHESS) facility at the Cornell High Energy Synchrotron Source (CHESS). CHESS is supported by the National Science Foundation (NSF) and NIGMS, National Institutes of Health (NIH) via NSF Award DMR-0225180, and the MacCHESS resource is supported by National Center for Research Resources (NCRR), NIH Award RR-01646.

      Supplementary Material

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