Materials and Methods
Protein expression and purification
Epsin 1 ENTH (1-164), and its mutants were made as N-terminal glutathione-S-transferase (GST) fusion proteins, being expressed in BL21 cells following IPTG-induction overnight at 22°C. Protein was purified from bacterial extracts by incubation with glutathione-sepharose beads, followed by extensive washing with 20mM HEPES, pH 7.4, 150mM NaCl, 1mM DTT. The protein was cleaved by incubation for 2 hours with thrombin. The cleaved product was purified further by passage over a Q-sepharose column. For crystallisation experiments, the protein was additionally passed down a Superdex200 gel-filtration column. For isothermal titration calorimetry experiments the proteins were dialysed into 50mM HEPES, pH 7.4, 120mM NaCl, 1mM DTT. cDNAs encoding full length rat epsin and the DPW domain (residues 249 to 401) were also expressed as GST fusion proteins (in pGex4T2) for bacterial expression and purified on Q-sepharose following thrombin cleavage. Myc-tagged proteins (in pCMV-myc) were used for COS-7 expression. Bacterial expression of the GST-tagged C-terminus of AP180 and the N-terminal domains of CALM and AP180 (residues 1-289, ANTH domain) and baculovirus expression and purification of full length AP180 were described in1. Eps15-C (residues 529-898) was expressed as a GST fusion protein. Clathrin was purified as previously described1, with modifications found at http://www2.mrc-lmb.cam.ac.uk/groups/hmm/techniqs/CCVprep.htm. This preparation does not exclude the presence of trace amounts of AP2 and AP180.
Liposome tubulation, sedimentation and electron microscopy
Synthetic liposomes contained 10% cholesterol, 40% phosphatidylethanolamine, 40% phosphatidylcholine, and 10% of the test lipid (in most cases phosphatidylinositol(4,5)bisphosphate). Bovine brain lipids (Folch Fraction 1, which contains approximately 10% phosphatidylinositol lipids) were purchased from Sigma (B1502). Liposomes were resuspended at 1mg/ml in 50mM HEPES, pH7.4, 120mM NaCl and extruded through a 0.4mm cyclopore filter. For electron microscopy (EM), proteins (4mM) were incubated with liposomes (0.1mg/ml) for 1min and absorbed onto a glow-discharged carbon-coated EM grid and stained with uranyl acetate before visualisation. For tubulation and sedimentation assays GST tags have been cleaved from the proteins but the presence of an N-terminal GST tag did not prevent either the tubulation of Folch liposomes by epsin or sedimentation. Hydrophobicity of the L6 mutants was determined using the Hopp-Woods scale2. For spin assays liposomes and protein (final volume of 40ml) were incubated for 10min at room temperature and then spun at 30,000rpm for 10min in a Beckman TLA 100 rotor.
Lipid monolayers (same composition as for synthetic liposomes) were formed on the surface of a buffer droplet in a Teflon block and protein(s) of interest were introduced into the buffer1. A carbon-coated gold EM grid was placed on the monolayer, incubated at room temperature for 60min, and then removed and stained with uranyl acetate.
COS cells were transfected using GeneJuice and 36 hours post transfection were incubated with biotinylated transferrin for 15min before fixation. Epsin transfected cells were detected using a polyclonal anti-Myc tag antibody (Cell Signalling, green in merged images), AP2 distribution was followed using an a-adaptin monoclonal (AP6, red in merged images) and biotinylated transferrin was detected with labelled avidin (blue in merged images). Transfections of RPMI cells were also performed using GeneJuice and up to 80% of cells were transfected. CCVs were purified as described previously3,4 and blotted for clathrin and AP2 adaptors.
Crystallisation and structure determination
Co-crystals of epsin ENTH (1-164) and Ins(1,4,5)P3 were grown by sitting drop vapour diffusion against a reservoir containing 35-38% dioxane, with a three-fold molar excess of Ins(1,4,5)P3 (Alexis Biochemicals)(approximately 1.8mM Ins(1,4,5)P3 against approximately 600mM epsin ENTH). Crystals, belonging to spacegroup I222 (a=41.21, b=95.82, c=118.15, a=b=g=90°), were obtained after 2-3 days. Crystals were cryoprotected by stepwise transfer to solutions containing 40% dioxane and 25% glycerol and X-ray diffraction data were collected on a rotating anode source (l = 1.54Å) and at beamline ID 29 at the European Synchrotron Radiation Facility, Grenoble, France (l = 0.98Å). Data were recorded using a Mar Research image plate (rotating anode) and an ADSC charge-coupled device detector. Data were integrated with MOSFLM5 and scaled using the CCP4 suite of programs6. Phases were obtained by molecular replacement, using AMoRe7, and using the published crystal structure of epsin ENTH8 (PDB 1EDU) as a search model. The resulting electron density map was excellent and extended to 1.7Å resolution. The model was built using O9 and was refined using Refmac510. Coordinates for the Ins(1,4,5)P3-epsin ENTH complex have been deposited in the PDB (ID 1H0A).
Isothermal titration calorimetry
Binding of inositol phosphates to epsin ENTH domain was investigated by isothermal titration calorimetry11 using a VP-ITC (MicroCal Inc., USA). All experiments were performed in 50mM HEPES, pH 7.4, 120mM NaCl, 1mM DTT at 10°C. The inositol phosphates were injected from a syringe in 30-40 steps up to a 3-4 fold molar excess. The cell contained 1.36ml protein solution and typically the inositol phosphate was added in steps of 5µl every 4min. Concentrations were chosen so that ENTH domains were at least 5 fold higher than the estimated dissociation constant, if possible. The inositol phosphates were again at least 10 fold more concentrated than the ENTH domains. The heat of dilution of the inositol phosphates was subtracted from the data prior to fitting. Titration curves were fitted to the data using the ORIGIN program supplied by the manufacturer yielding the stoichiometry N, the binary equilibrium constant Ka (= Kd-1) and the enthalpy of binding (Supplementary Table 2). The entropy of binding DS° was calculated from the relationship DG°=-RT lnKa and the Gibbs-Helmholtz equation.
1. Ford, M. G. J. et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 291, 1051-1055 (2001).
2. Hopp, T. P. & Woods, K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl Acad. Sci. USA 78, 3824-3828 (1981).
3. Pearse, B. M. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc. Natl Acad. Sci. USA 79, 451-455 (1982).
4. Metzler, M. et al. HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J. Biol. Chem. 276, 39271-39276. (2001).
5. Leslie, A. G. W. in Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography No. 26 (SERC, Daresbury Laboratory, Warrington, UK, 1992).
6. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Cryst. D50, 760-763 (1994).
7. Navaza, J. AMORE-an automated package for molecular replacement. Acta Cryst. A50, 157-163 (1994).
8. Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P. & Brunger, A. T. Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). J. Cell Biol. 149, 537-546 (2000).
9. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst. A47, 110-119 (1991).
10. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Cryst. D53, 240-255 (1997).
11. Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131-137 (1989).