PDBsum entry 2qpt

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Endocytosis PDB id
Jmol PyMol
Protein chain
476 a.a. *
Waters ×6
* Residue conservation analysis
PDB id:
Name: Endocytosis
Title: Crystal structure of an ehd atpase involved in membrane remo
Structure: Eh domain-containing protein-2. Chain: a. Engineered: yes. Mutation: yes
Source: Mus musculus. House mouse. Organism_taxid: 10090. Gene: ehd2. Expressed in: escherichia coli. Expression_system_taxid: 562.
3.10Å     R-factor:   0.236     R-free:   0.276
Authors: O.Daumke
Key ref:
O.Daumke et al. (2007). Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling. Nature, 449, 923-927. PubMed id: 17914359 DOI: 10.1038/nature06173
25-Jul-07     Release date:   16-Oct-07    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q8BH64  (EHD2_MOUSE) -  EH domain-containing protein 2
543 a.a.
476 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   13 terms 
  Biological process     protein localization in plasma membrane   8 terms 
  Biochemical function     nucleotide binding     8 terms  


DOI no: 10.1038/nature06173 Nature 449:923-927 (2007)
PubMed id: 17914359  
Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling.
O.Daumke, R.Lundmark, Y.Vallis, S.Martens, P.J.Butler, H.T.McMahon.
The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.
  Selected figure(s)  
Figure 1.
Figure 1: EHD2 shares common properties with the dynamin superfamily. a, Domain structure of EHD proteins (numbering from mouse EHD2 amino acids). b, EHD2 binds to adenine nucleotides, as determined by isothermal titration calorimetry. For clarity, H = H[n] - H[1] is plotted. c, Coomassie-stained gels of liposome co-sedimentation assays in the presence of 1 mM ATP- -S using 0.8- m-filtered Folch, 100% PtdSer or synthetic liposomes containing 10% of the indicated PtdIns (plus 70% Ptd choline, 10% PtdSer, 10% cholesterol). S, supernatant; P, pelleted fraction. d, Electron micrographs of negatively stained PtdSer liposomes in the absence (left panel) or presence (middle, right panels) of EHD2 and 1 mM ATP- -S. The right panel shows an intermediate in the tubulation process, surrounded by EHD2 rings of variable diameter. e, Amino-terminally EGFP-tagged EHD2 wild type and the T72A mutant were overexpressed in HeLa cells. Confocal images were acquired close to the basal cell surface; the scale bar is 10 m. f, Nucleotide hydrolysis by EHD2 was measured by HPLC. Intrinsic reactions in absence of lipids are open symbols (mean s.d., n = 2) and stimulated reactions are filled symbols (mean s.d., n = 3). Whereas the intrinsic ATP reaction was eightfold-stimulated by Folch lipids (open versus filled circles), GTP hydrolysis was not stimulated (open versus filled triangles). The nucleotide-free T72A mutant did not show stimulation of ATP hydrolysis (open versus filled squares).
Figure 3.
Figure 3: Membrane binding and the role of ATP hydrolysis. a, Putative membrane-binding site with residues tested for membrane binding in ball-and-stick representation. The highly curved membrane interaction site of EHD2 is indicated. b, Sedimentation assays in the presence (upper panel) and absence (lower panel) of Folch liposomes using wild-type EHD2 and mutants, as in Fig. 1c. c, Nucleotide hydrolysis of lipid binding mutants as described in Fig. 1f. The wild-type protein had a k[obs] of 1.6 h^-1 (open circles, intrinsic; filled circles, lipid stimulated). The F322A mutant (open versus filled inverted triangles) showed a 40% decrease in the stimulated ATPase reaction (k[obs] = 3.0 h^-1), and the K328D mutant (open versus filled triangles) showed a 75% reduced rate (k[obs] = 1.6 h^-1), whereas for the K327D mutant (open versus filled diamonds) stimulation was barely visible. d, EGFP-tagged F322A mutant showed a completely cytoplasmic distribution when overexpressed in HeLa cells. Scale bar, 10 m. e, Affinity to ATP- -S was measured as in Fig. 1b. Nucleotide hydrolysis was measured as described in Fig. 1f (values represent mean s.e.m.; n = 2, intrinsic; n = 3, stimulated reactions). In vitro tubulation activity of PtdSer liposomes was analysed as described in Fig. 1d. f, Confocal images of HeLa cells overexpressing the indicated mutants. Scale bar, 10 m. g, Quantification of the overexpression phenotypes from Fig. 3f. For each construct, three independent experiments with 50 cells per experiment were analysed.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2007, 449, 923-927) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
23340574 R.G.Parton, and M.A.Del Pozo (2013).
Caveolae as plasma membrane sensors, protectors and organizers.
  Nat Rev Mol Cell Biol, 14, 98.  
22193161 P.J.Cullen, and H.C.Korswagen (2012).
Sorting nexins provide diversity for retromer-dependent trafficking events.
  Nat Cell Biol, 14, 29-37.  
22233676 S.M.Ferguson, and P.De Camilli (2012).
Dynamin, a membrane-remodelling GTPase.
  Nat Rev Mol Cell Biol, 13, 75-88.  
20961375 B.Cai, D.Katafiasz, V.Horejsi, and N.Naslavsky (2011).
Pre-sorting endosomal transport of the GPI-anchored protein, CD59, is regulated by EHD1.
  Traffic, 12, 102-120.  
21067929 N.Naslavsky, and S.Caplan (2011).
EHD proteins: key conductors of endocytic transport.
  Trends Cell Biol, 21, 122-131.  
21276251 N.Pawlowski, A.Khaminets, J.P.Hunn, N.Papic, A.Schmidt, R.C.Uthaiah, R.Lange, G.Vopper, S.Martens, E.Wolf, and J.C.Howard (2011).
The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii.
  BMC Biol, 9, 7.  
21278333 O.Daumke, and G.J.Praefcke (2011).
Structural insights into membrane fusion at the endoplasmic reticulum.
  Proc Natl Acad Sci U S A, 108, 2175-2176.  
21187387 P.Philippidou, G.Valdez, W.Akmentin, W.J.Bowers, H.J.Federoff, and S.Halegoua (2011).
Trk retrograde signaling requires persistent, Pincher-directed endosomes.
  Proc Natl Acad Sci U S A, 108, 852-857.  
21219150 T.Baumgart, B.R.Capraro, C.Zhu, and S.L.Das (2011).
Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids.
  Annu Rev Phys Chem, 62, 483-506.  
20083601 A.Joset, D.A.Dodd, S.Halegoua, and M.E.Schwab (2010).
Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling.
  J Cell Biol, 188, 271-285.  
20573983 A.Shi, C.C.Chen, R.Banerjee, D.Glodowski, A.Audhya, C.Rongo, and B.D.Grant (2010).
EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans.
  Mol Biol Cell, 21, 2930-2943.  
21170358 B.Chen, Y.Jiang, S.Zeng, J.Yan, X.Li, Y.Zhang, W.Zou, and X.Wang (2010).
Endocytic sorting and recycling require membrane phosphatidylserine asymmetry maintained by TAT-1/CHAT-1.
  PLoS Genet, 6, e1001235.  
20463227 C.C.Yap, Z.M.Lasiecka, S.Caplan, and B.Winckler (2010).
Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting.
  J Neurosci, 30, 6646-6657.  
19965594 C.M.Blouin, S.Le Lay, A.Eberl, H.C.Köfeler, I.C.Guerrera, C.Klein, X.Le Liepvre, F.Lasnier, O.Bourron, J.F.Gautier, P.Ferré, E.Hajduch, and I.Dugail (2010).
Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects.
  J Lipid Res, 51, 945-956.  
20305790 D.Rapaport, Y.Lugassy, E.Sprecher, and M.Horowitz (2010).
Loss of SNAP29 impairs endocytic recycling and cell motility.
  PLoS One, 5, e9759.  
21059949 D.Schwefel, C.Fröhlich, J.Eichhorst, B.Wiesner, J.Behlke, L.Aravind, and O.Daumke (2010).
Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2).
  Proc Natl Acad Sci U S A, 107, 20299-20304.
PDB codes: 2xtm 2xtn 2xto 2xtp
20329706 G.D.Henry, D.J.Corrigan, J.V.Dineen, and J.D.Baleja (2010).
Charge effects in the selection of NPF motifs by the EH domain of EHD1.
  Biochemistry, 49, 3381-3392.  
21115825 G.Ko, S.Paradise, H.Chen, M.Graham, M.Vecchi, F.Bianchi, O.Cremona, P.P.Di Fiore, and P.De Camilli (2010).
Selective high-level expression of epsin 3 in gastric parietal cells, where it is localized at endocytic sites of apical canaliculi.
  Proc Natl Acad Sci U S A, 107, 21511-21516.  
19914387 J.R.van Weering, P.Verkade, and P.J.Cullen (2010).
SNX-BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting.
  Semin Cell Dev Biol, 21, 371-380.  
19932206 K.Rochlin, S.Yu, S.Roy, and M.K.Baylies (2010).
Myoblast fusion: when it takes more to make one.
  Dev Biol, 341, 66-83.  
20359371 M.A.Rainey, M.George, G.Ying, R.Akakura, D.J.Burgess, E.Siefker, T.Bargar, L.Doglio, S.E.Crawford, G.L.Todd, V.Govindarajan, R.A.Hess, V.Band, M.Naramura, and H.Band (2010).
The endocytic recycling regulator EHD1 is essential for spermatogenesis and male fertility in mice.
  BMC Dev Biol, 10, 37.  
21054155 M.Anitei, T.Wassmer, C.Stange, and B.Hoflack (2010).
Bidirectional transport between the trans-Golgi network and the endosomal system.
  Mol Membr Biol, 27, 443-456.  
  20213691 M.George, M.A.Rainey, M.Naramura, G.Ying, D.W.Harms, M.H.Vitaterna, L.Doglio, S.E.Crawford, R.A.Hess, V.Band, and H.Band (2010).
Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice.
  Genesis, 48, 328-342.  
20638285 M.M.Kozlov, H.T.McMahon, and L.V.Chernomordik (2010).
Protein-driven membrane stresses in fusion and fission.
  Trends Biochem Sci, 35, 699-706.  
  20585517 M.Sharma, S.S.Giridharan, J.Rahajeng, S.Caplan, and N.Naslavsky (2010).
MICAL-L1: An unusual Rab effector that links EHD1 to tubular recycling endosomes.
  Commun Integr Biol, 3, 181-183.  
20159556 P.D.Allaire, A.L.Marat, C.Dall'Armi, G.Di Paolo, P.S.McPherson, and B.Ritter (2010).
The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.
  Mol Cell, 37, 370-382.  
20427576 P.Verma, A.G.Ostermeyer-Fay, and D.A.Brown (2010).
Caveolin-1 induces formation of membrane tubules that sense actomyosin tension and are inhibited by polymerase I and transcript release factor/cavin-1.
  Mol Biol Cell, 21, 2226-2240.  
19931628 R.Lundmark, and S.R.Carlsson (2010).
Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis.
  Semin Cell Dev Biol, 21, 363-370.  
20428112 S.Gao, A.von der Malsburg, S.Paeschke, J.Behlke, O.Haller, G.Kochs, and O.Daumke (2010).
Structural basis of oligomerization in the stalk region of dynamin-like MxA.
  Nature, 465, 502-506.
PDB code: 3ljb
19696797 B.D.Grant, and J.G.Donaldson (2009).
Pathways and mechanisms of endocytic recycling.
  Nat Rev Mol Cell Biol, 10, 597-608.  
19765184 E.L.Clayton, and M.A.Cousin (2009).
The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles.
  J Neurochem, 111, 901-914.  
19568784 F.Campelo (2009).
Modeling morphological instabilities in lipid membranes with anchored amphiphilic polymers.
  J Chem Biol, 2, 65-80.  
19798736 F.Kieken, M.Jović, M.Tonelli, N.Naslavsky, S.Caplan, and P.L.Sorgen (2009).
Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1.
  Protein Sci, 18, 2471-2479.
PDB codes: 2kff 2kfg 2kfh
20064379 H.H.Low, C.Sachse, L.A.Amos, and J.Löwe (2009).
Structure of a bacterial dynamin-like protein lipid tube provides a mechanism for assembly and membrane curving.
  Cell, 139, 1342-1352.
PDB code: 2w6d
19150238 J.Saarikangas, H.Zhao, A.Pykäläinen, P.Laurinmäki, P.K.Mattila, P.K.Kinnunen, S.J.Butcher, and P.Lappalainen (2009).
Molecular mechanisms of membrane deformation by I-BAR domain proteins.
  Curr Biol, 19, 95.  
19936242 M.Bar, M.Sharfman, S.Schuster, and A.Avni (2009).
The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling.
  PLoS One, 4, e7973.  
19369419 M.Jović, F.Kieken, N.Naslavsky, P.L.Sorgen, and S.Caplan (2009).
Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling.
  Mol Biol Cell, 20, 2731-2743.  
  19907710 M.Sharma, M.Jovic, F.Kieken, N.Naslavsky, P.Sorgen, and S.Caplan (2009).
A model for the role of EHD1-containing membrane tubules in endocytic recycling.
  Commun Integr Biol, 2, 431-433.  
19864458 M.Sharma, S.S.Giridharan, J.Rahajeng, N.Naslavsky, and S.Caplan (2009).
MICAL-L1 links EHD1 to tubular recycling endosomes and regulates receptor recycling.
  Mol Biol Cell, 20, 5181-5194.  
19139087 N.Naslavsky, J.McKenzie, N.Altan-Bonnet, D.Sheff, and S.Caplan (2009).
EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology.
  J Cell Sci, 122, 389-400.  
19814798 P.M.van Bergen En Henegouwen (2009).
Eps15: a multifunctional adaptor protein regulating intracellular trafficking.
  Cell Commun Signal, 7, 24.  
19494235 P.N.Bernatchez, A.Sharma, P.Kodaman, and W.C.Sessa (2009).
Myoferlin is critical for endocytosis in endothelial cells.
  Am J Physiol Cell Physiol, 297, C484-C492.  
19915558 S.Pant, M.Sharma, K.Patel, S.Caplan, C.M.Carr, and B.D.Grant (2009).
AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling.
  Nat Cell Biol, 11, 1399-1410.  
19619496 T.Wassmer, N.Attar, M.Harterink, J.R.van Weering, C.J.Traer, J.Oakley, B.Goud, D.J.Stephens, P.Verkade, H.C.Korswagen, and P.J.Cullen (2009).
The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.
  Dev Cell, 17, 110-122.  
18801062 B.D.Grant, and S.Caplan (2008).
Mechanisms of EHD/RME-1 protein function in endocytic transport.
  Traffic, 9, 2043-2052.  
18515373 F.Campelo, H.T.McMahon, and M.M.Kozlov (2008).
The hydrophobic insertion mechanism of membrane curvature generation by proteins.
  Biophys J, 95, 2325-2339.  
18200045 J.Rumpf, B.Simon, N.Jung, T.Maritzen, V.Haucke, M.Sattler, and Y.Groemping (2008).
Structure of the Eps15-stonin2 complex provides a molecular explanation for EH-domain ligand specificity.
  EMBO J, 27, 558-569.
PDB code: 2jxc
18472259 J.S.Bonifacino, and J.H.Hurley (2008).
  Curr Opin Cell Biol, 20, 427-436.  
18502764 K.R.Doherty, A.R.Demonbreun, G.Q.Wallace, A.Cave, A.D.Posey, K.Heretis, P.Pytel, and E.M.McNally (2008).
The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion.
  J Biol Chem, 283, 20252-20260.  
18331452 M.Sharma, N.Naslavsky, and S.Caplan (2008).
A role for EHD4 in the regulation of early endosomal transport.
  Traffic, 9, 995.  
18502633 P.G.Woodman, and C.E.Futter (2008).
Multivesicular bodies: co-ordinated progression to maturity.
  Curr Opin Cell Biol, 20, 408-414.  
18689681 R.Beck, Z.Sun, F.Adolf, C.Rutz, J.Bassler, K.Wild, I.Sinning, E.Hurt, B.Brügger, J.Béthune, and F.Wieland (2008).
Membrane curvature induced by Arf1-GTP is essential for vesicle formation.
  Proc Natl Acad Sci U S A, 105, 11731-11736.  
18442980 Y.Shibata, C.Voss, J.M.Rist, J.Hu, T.A.Rapoport, W.A.Prinz, and G.K.Voeltz (2008).
The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum.
  J Biol Chem, 283, 18892-18904.  
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.