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PDBsum entry 3b9p

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protein metals links
Hydrolase PDB id
3b9p
Jmol
Contents
Protein chain
268 a.a. *
Metals
_CL
Waters ×15
* Residue conservation analysis
PDB id:
3b9p
Name: Hydrolase
Title: Spastin
Structure: Cg5977-pa, isoform a. Chain: a. Fragment: residues 463-758. Synonym: cg5977-pb, isoform b. Engineered: yes
Source: Drosophila melanogaster. Fruit fly. Organism_taxid: 7227. Gene: spas. Expressed in: escherichia coli. Expression_system_taxid: 562
Resolution:
2.70Å     R-factor:   0.245     R-free:   0.287
Authors: A.Roll-Mecak,R.D.Vale
Key ref:
A.Roll-Mecak and R.D.Vale (2008). Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature, 451, 363-367. PubMed id: 18202664 DOI: 10.1038/nature06482
Date:
06-Nov-07     Release date:   22-Jan-08    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q8I0P1  (SPAST_DROME) -  Spastin
Seq:
Struc:
 
Seq:
Struc:
758 a.a.
268 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.3.6.4.3  - Microtubule-severing ATPase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP + H2O = ADP + phosphate
ATP
+ H(2)O
= ADP
+ phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biochemical function     nucleotide binding     3 terms  

 

 
    reference    
 
 
DOI no: 10.1038/nature06482 Nature 451:363-367 (2008)
PubMed id: 18202664  
 
 
Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin.
A.Roll-Mecak, R.D.Vale.
 
  ABSTRACT  
 
Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.
 
  Selected figure(s)  
 
Figure 1.
Figure 1: X-ray structure of the nucleotide-free AAA domain of spastin. a, Domain structure of Drosophila spastin: grey, N-terminal domain; red, linker (exon 4, absent in the shorter isoform of spastin used in this study, is hatched); and the AAA domain (coloured according to the X-ray structure). NBD, nucleotide-binding domain; HBD, four-helix bundle domain. Two potential start codons (ATG) are shown (see Supplementary Methods for discussion). The N-terminal boundary of the AAA domain is based on our X-ray structure and differs from that of ref. 14. A segment of the structurally important N-terminal helix of the AAA domain is within what the authors of ref. 14 define as a microtubule-binding domain. The MIT + AAA and AAA constructs are shown schematically below. b, Left, MIT + AAA disassembles the microtubule network when transfected in Drosophila S2 cells and when added to microtubules in vitro, but AAA has no detectable activity at the same concentration (0.15 M). (Weak severing is observed at higher concentrations, Supplementary Fig. 1.) Arrows indicate breaks in microtubules. Scale bar, 5 m. Right, microtubule (MT)-binding and ATPase activities of MIT + AAA and AAA. Microtubule-binding affinity was determined for the Walker B E583Q mutant, which is a stable hexamer and is inactive in severing. c, Ribbon representation of the spastin AAA domain crystal structure. N-terminal helix/loop, magenta; NBD, light green; HBD, dark green; C-terminal helix, blue. The pink sphere depicts a chloride ion. d, Conserved hydrophobic interactions between the N-terminal helix and the main body of the NBD. e, Conserved interactions between the C-terminal helix and the P loop. f, ATPase (red) and microtubule-severing (blue) rates of N- and C-terminal helix mutants. Error bars represent standard errors of the mean (see Methods). WT, wild type. g, Detail of the superposition of spastin and ATP-bound NSF structures^15, showing contacts that keep the N-terminal flap of monomeric spastin (magenta) in an open conformation, unable to stabilize the nucleotide or interact with the neighbouring protomer. Spastin is colour-coded as in panel c. NSF is in grey. Dashed lines, hydrogen bonds.
Figure 4.
Figure 4: Proposed mechanism of severing by spastin and effects of disease mutations. a, Proposed mechanism for microtubule-severing by spastin. The spastin AAA core is shown in cyan with pore loops 1, 2 and 3 highlighted in red and numbered in the figure. The MIT domains are shown as gold ovals. The valency of the interaction of the MIT domains with the microtubule is unknown. On the basis of affinity measurements, it is likely that not all MIT domains are engaged with the microtubule (the potentially unengaged MIT domain is shown hatched). The tubulin heterodimers forming the microtubule are shown in green as a ribbon representation, whereas the C-terminal tubulin tails are shown in red cartoon representation. b, Left, molecular surface of spastin (face A). One protomer is shown in a ribbon representation and residues mutated in HSP patients are shown as violet spheres. Right, in addition to mapping to the pore loops (S589Y, R601L, P631L), disease mutations can interfere with ATP binding (F522C, N527K, K529R) and protomer–protomer interactions (D697N, R704Q, R641C, R601L, P631L). G511R maps to a loop on face A where it could destabilize protomer–protomer interactions and/or the microtubule-binding interface (Supplementary Fig. 4).
 
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 451, 363-367) copyright 2008.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21436431 C.Raiborg, and H.Stenmark (2011).
Cell biology. A helix for the final cut.
  Science, 331, 1533-1534.  
20665701 J.C.Fuerst, A.W.Henkel, A.Stroebel, O.Welzel, T.W.Groemer, J.Kornhuber, and D.Bönsch (2011).
Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations.
  J Cell Physiol, 226, 362-368.  
22056769 M.Stotz, O.Mueller-Cajar, S.Ciniawsky, P.Wendler, F.U.Hartl, A.Bracher, and M.Hayer-Hartl (2011).
Structure of green-type Rubisco activase from tobacco.
  Nat Struct Mol Biol, 18, 1366-1370.
PDB codes: 3t15 3zw6
22048315 O.Mueller-Cajar, M.Stotz, P.Wendler, F.U.Hartl, A.Bracher, and M.Hayer-Hartl (2011).
Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.
  Nature, 479, 194-199.
PDB codes: 3syk 3syl 3zuh
21549946 P.W.Baas, and V.Sharma (2011).
Cell migration: katanin gives microtubules a trim.
  Curr Biol, 21, R302-R304.  
20857310 S.Miura, H.Shibata, H.Kida, K.Noda, T.Toyama, N.Iwasaki, A.Iwaki, M.Ayabe, H.Aizawa, T.Taniwaki, and Y.Fukumaki (2011).
Partial SPAST and DPY30 deletions in a Japanese spastic paraplegia type 4 family.
  Neurogenetics, 12, 25-31.  
19963362 A.Roll-Mecak, and F.J.McNally (2010).
Microtubule-severing enzymes.
  Curr Opin Cell Biol, 22, 96.  
20154147 B.Gilquin, E.Taillebourg, N.Cherradi, A.Hubstenberger, O.Gay, N.Merle, N.Assard, M.O.Fauvarque, S.Tomohiro, O.Kuge, and J.Baudier (2010).
The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes.
  Mol Cell Biol, 30, 1984-1996.  
20530212 B.Lacroix, J.van Dijk, N.D.Gold, J.Guizetti, G.Aldrian-Herrada, K.Rogowski, D.W.Gerlich, and C.Janke (2010).
Tubulin polyglutamylation stimulates spastin-mediated microtubule severing.
  J Cell Biol, 189, 945-954.  
19700636 D.Wloga, D.Dave, J.Meagley, K.Rogowski, M.Jerka-Dziadosz, and J.Gaertig (2010).
Hyperglutamylation of tubulin can either stabilize or destabilize microtubules in the same cell.
  Eukaryot Cell, 9, 184-193.  
20154342 F.Du, E.F.Ozdowski, I.K.Kotowski, D.A.Marchuk, and N.T.Sherwood (2010).
Functional conservation of human Spastin in a Drosophila model of autosomal dominant-hereditary spastic paraplegia.
  Hum Mol Genet, 19, 1883-1896.  
20159162 H.Joshi, F.Momin, K.E.Haines, and R.I.Dima (2010).
Exploring the contribution of collective motions to the dynamics of forced-unfolding in tubulin.
  Biophys J, 98, 657-666.  
20430936 J.M.Solowska, J.Y.Garbern, and P.W.Baas (2010).
Evaluation of loss of function as an explanation for SPG4-based hereditary spastic paraplegia.
  Hum Mol Genet, 19, 2767-2779.  
20365773 S.H.Tindemans, and B.M.Mulder (2010).
Microtubule length distributions in the presence of protein-induced severing.
  Phys Rev E Stat Nonlin Soft Matter Phys, 81, 031910.  
20022957 S.Sugimoto, K.Yamanaka, S.Nishikori, A.Miyagi, T.Ando, and T.Ogura (2010).
AAA+ chaperone ClpX regulates dynamics of prokaryotic cytoskeletal protein FtsZ.
  J Biol Chem, 285, 6648-6657.  
19535732 B.McDonald, and J.Martin-Serrano (2009).
No strings attached: the ESCRT machinery in viral budding and cytokinesis.
  J Cell Sci, 122, 2167-2177.  
19131969 G.Bönemann, A.Pietrosiuk, A.Diemand, H.Zentgraf, and A.Mogk (2009).
Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.
  EMBO J, 28, 315-325.  
19541655 J.L.Camberg, J.R.Hoskins, and S.Wickner (2009).
ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.
  Proc Natl Acad Sci U S A, 106, 10614-10619.  
19000169 J.W.Connell, C.Lindon, J.P.Luzio, and E.Reid (2009).
Spastin couples microtubule severing to membrane traffic in completion of cytokinesis and secretion.
  Traffic, 10, 42-56.  
19278657 M.J.Landsberg, P.R.Vajjhala, R.Rothnagel, A.L.Munn, and B.Hankamer (2009).
Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding.
  Structure, 17, 427-437.  
19896110 M.R.Abdollahi, E.Morrison, T.Sirey, Z.Molnar, B.E.Hayward, I.M.Carr, K.Springell, C.G.Woods, M.Ahmed, L.Hattingh, P.Corry, D.T.Pilz, N.Stoodley, Y.Crow, G.R.Taylor, D.T.Bonthron, and E.Sheridan (2009).
Mutation of the variant alpha-tubulin TUBA8 results in polymicrogyria with optic nerve hypoplasia.
  Am J Hum Genet, 85, 737-744.  
18849407 R.G.Coleman, and K.A.Sharp (2009).
Finding and characterizing tunnels in macromolecules with application to ion channels and pores.
  Biophys J, 96, 632-645.  
18606141 C.Kieffer, J.J.Skalicky, E.Morita, I.De Domenico, D.M.Ward, J.Kaplan, and W.I.Sundquist (2008).
Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal protein targeting and HIV-1 budding.
  Dev Cell, 15, 62-73.
PDB code: 2k3w
18410514 D.V.Pantakani, L.S.Swapna, N.Srinivasan, and A.U.Mannan (2008).
Spastin oligomerizes into a hexamer and the mutant spastin (E442Q) redistribute the wild-type spastin into filamentous microtubule.
  J Neurochem, 106, 613-624.  
18997780 D.Yang, N.Rismanchi, B.Renvoisé, J.Lippincott-Schwartz, C.Blackstone, and J.H.Hurley (2008).
Structural basis for midbody targeting of spastin by the ESCRT-III protein CHMP1B.
  Nat Struct Mol Biol, 15, 1278-1286.
PDB code: 3eab
18541115 G.Stevanin, M.Ruberg, and A.Brice (2008).
Recent advances in the genetics of spastic paraplegias.
  Curr Neurol Neurosci Rep, 8, 198-210.  
18929572 M.D.Gonciarz, F.G.Whitby, D.M.Eckert, C.Kieffer, A.Heroux, W.I.Sundquist, and C.P.Hill (2008).
Biochemical and structural studies of yeast Vps4 oligomerization.
  J Mol Biol, 384, 878-895.
PDB codes: 3eie 3eih
18840679 R.I.Dima, and H.Joshi (2008).
Probing the origin of tubulin rigidity with molecular simulations.
  Proc Natl Acad Sci U S A, 105, 15743-15748.  
19033201 T.K.Rostovtseva, K.L.Sheldon, E.Hassanzadeh, C.Monge, V.Saks, S.M.Bezrukov, and D.L.Sackett (2008).
Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
  Proc Natl Acad Sci U S A, 105, 18746-18751.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.