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• W1969667037 abstract "Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and β-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the α-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the α-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the α-appendage. Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and β-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the α-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the α-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the α-appendage. Clathrin-mediated endocytosis is the process whereby proteins and lipids destined for internalization from the plasma membrane are packaged into vesicles with the aid of a clathrin coat. Purified coated vesicles from brain contain three major components as follows: clathrin, AP180, and AP2 complexes (1Pearse B.M. Robinson M.S. EMBO J. 1984; 3: 1951-1957Crossref PubMed Scopus (148) Google Scholar, 2Ahle S. Ungewickell E. EMBO J. 1986; 5: 3143-3149Crossref PubMed Scopus (144) Google Scholar, 3Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (605) Google Scholar). Clathrin triskelia oligomerize to provide the scaffold around the forming vesicle (and can form similar cages in solution (4Fotin A. Cheng Y. Sliz P. Grigorieff N. Harrison S.C. Kirchhausen T. Walz T. Nature. 2004; 432: 573-579Crossref PubMed Scopus (408) Google Scholar)). With its terminal domain, clathrin interacts with other endocytic proteins, including the AP2 7The abbreviations used are: AP2adaptor protein complex 2AmphamphiphysinAP180adaptor protein of 180 kDaBARBin2/amphiphysin/RvsCALMclathrin assembly lymphoid myeloid leukemia proteinDab2Disabled 2Eps15epidermal growth factor receptor pathway substrate 15epsinEps15-interacting proteinGSTglutathione S-transferaseHIP1Huntingtin interacting protein 1ITCisothermal titration calorimetryPLAAphospholipase A2 activatorPDBProtein Data BankSHSrc homologyDTTdithiothreitolPtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphate. complex, AP180, epsin, disabled-2 (Dab2), and amphiphysin. These interactions are mediated via short motifs; for example, clathrin binds to amphiphysin through motifs such as LLDLD or PWXXW (5Miele A.E. Watson P.J. Evans P.R. Traub L.M. Owen D.J. Nat. Struct. Mol. Biol. 2004; 11: 242-248Crossref PubMed Scopus (96) Google Scholar, 6Dell'Angelica E.C. Trends Cell Biol. 2001; 11: 315-318Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Because oligomeric clathrin presents an array of binding sites for these motifs, it serves as a network hub, organizing binding partners within the lattice. The AP complexes, as well as many accessory proteins and alternative cargo adaptors such as AP180, Dab2, epsin, and amphiphysin, recruit clathrin to PtdIns(4,5)P2-rich areas in the membrane and promote its polymerization into a lattice. Because of its significant number of interaction partners, another key hub in the endocytic interactome is the AP2 complex (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar, 8Schmid E.M. Ford M.G. Burtey A. Praefcke G.J. Peak-Chew S.Y. Mills I.G. Benmerah A. McMahon H.T. PLoS Biol. 2006; 4: e262Crossref PubMed Scopus (193) Google Scholar, 9Schmid E.M. McMahon H.T. Nature. 2007; 448: 883-888Crossref PubMed Scopus (253) Google Scholar). It consists of four subunits (α, β2, μ2, and σ2) and forms a stable heterotetramer in solution (10Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Using electron microscopy it was shown that the AP2 complex can be subdivided into the following: (i) a trunk domain, which interacts with cargo proteins and PtdIns(4,5)P2, and (ii) two appendage domains made from the C termini of the α- and β-subunits, which interact with a large number of accessory proteins by binding to short motifs in these proteins. For example, the α-adaptin appendage binds to DX(F/W), FXDXF, WXX(F/W), and FXXFXXL motifs (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar, 11Mills I.G. Praefcke G.J. Vallis Y. Peter B.J. Olesen L.E. Gallop J.L. Butler P.J. Evans P.R. McMahon H.T. J. Cell Biol. 2003; 160: 213-222Crossref PubMed Scopus (203) Google Scholar, 12Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 13Mishra S.K. Hawryluk M.J. Brett T.J. Keyel P.A. Dupin A.L. Jha A. Heuser J.E. Fremont D.H. Traub L.M. J. Biol. Chem. 2004; 279: 46191-46203Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Mattera R. Ritter B. Sidhu S.S. McPherson P.S. Bonifacino J.S. J. Biol. Chem. 2004; 279: 8018-8028Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 15Ritter B. Philie J. Girard M. Tung E.C. Blondeau F. McPherson P.S. EMBO Rep. 2003; 4: 1089-1095Crossref PubMed Scopus (67) Google Scholar, 16Wasiak S. Denisov A.Y. Han Z. Leventis P.A. de Heuvel E. Boulianne G.L. Kay B.K. Gehring K. McPherson P.S. FEBS Lett. 2003; 555: 437-442Crossref PubMed Scopus (16) Google Scholar). These can be highly clustered in motif domains of the accessory proteins where they are also frequently found close to clathrin-binding motifs. The appendage domains are connected to the trunk domain by flexible linkers, which lack a defined secondary structure in solution. adaptor protein complex 2 amphiphysin adaptor protein of 180 kDa Bin2/amphiphysin/Rvs clathrin assembly lymphoid myeloid leukemia protein Disabled 2 epidermal growth factor receptor pathway substrate 15 Eps15-interacting protein glutathione S-transferase Huntingtin interacting protein 1 isothermal titration calorimetry phospholipase A2 activator Protein Data Bank Src homology dithiothreitol phosphatidylinositol 4,5-bisphosphate. Although the AP2 appendages are only 16% identical in terms of their sequences, they are structurally very similar (12Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 17Owen D.J. Vallis Y. Pearse B.M. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar). We and others have previously proposed that both the α- and β2-appendage domains bind to DX(F/W) motifs in accessory proteins (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar, 8Schmid E.M. Ford M.G. Burtey A. Praefcke G.J. Peak-Chew S.Y. Mills I.G. Benmerah A. McMahon H.T. PLoS Biol. 2006; 4: e262Crossref PubMed Scopus (193) Google Scholar, 12Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 18Brett T.J. Traub L.M. Fremont D.H. Structure (Lond.). 2002; 10: 797-809Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The DX(F/W) motifs on accessory proteins are often found in proline-rich regions. For example, rat epsin1 has nine DPW motifs, and the majority of these are found in a proline-rich stretch of 105 amino acids, a region that by CD spectroscopy has no obvious structure (19Kalthoff C. Alves J. Urbanke C. Knorr R. Ungewickell E.J. J. Biol. Chem. 2002; 277: 8209-8216Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Human Eps15 contains 15 DPF and two other DXF motifs in a stretch of 230 amino acids. These motifs may allow clustering of the AP2 complexes at the endocytic assembly zones and enhance the binding to PtdIns(4,5)P2-containing membranes (8Schmid E.M. Ford M.G. Burtey A. Praefcke G.J. Peak-Chew S.Y. Mills I.G. Benmerah A. McMahon H.T. PLoS Biol. 2006; 4: e262Crossref PubMed Scopus (193) Google Scholar, 20Hinrichsen L. Meyerholz A. Groos S. Ungewickell E.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8715-8720Crossref PubMed Scopus (91) Google Scholar). DX(F/W) motifs bind to sites on the platform subdomains of the appendages, centered around a hydrophobic pocket (Trp-840 in α; Trp-841 in β2) (12Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 17Owen D.J. Vallis Y. Pearse B.M. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar) in a tight turn conformation (18Brett T.J. Traub L.M. Fremont D.H. Structure (Lond.). 2002; 10: 797-809Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In the study by Brett et al. (18Brett T.J. Traub L.M. Fremont D.H. Structure (Lond.). 2002; 10: 797-809Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), the authors also found a secondary binding site for DPW motifs on the β-sandwich subdomain of the α-appendage. More importantly, they found that an FEDNF peptide from amphiphysin binds to the top of the α-appendage using the site around Trp-840 but in a different conformation by inserting the first Phe residue into the hydrophobic pocket. It was therefore proposed that FXDXF is a general high affinity binding motif for the α-adaptin appendage. Recently, a fourth binding motif for the α-adaptin appendage has been identified in the proteins NECAP, stonin, synaptojanin, connecdenn, and others (15Ritter B. Philie J. Girard M. Tung E.C. Blondeau F. McPherson P.S. EMBO Rep. 2003; 4: 1089-1095Crossref PubMed Scopus (67) Google Scholar, 21Allaire P.D. Ritter B. Thomas S. Burman J.L. Denisov A.Y. Legendre-Guillemin V. Harper S.Q. Davidson B.L. Gehring K. McPherson P.S. J. Neurosci. 2006; 26: 13202-13212Crossref PubMed Scopus (54) Google Scholar, 22Jha A. Agostinelli N.R. Mishra S.K. Keyel P.A. Hawryluk M.J. Traub L.M. J. Biol. Chem. 2004; 279: 2281-2290Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Walther K. Diril M.K. Jung N. Haucke V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 964-969Crossref PubMed Scopus (79) Google Scholar). The binding motif consensus WXX(F/W) resembles the second clathrin-binding motif PWXXW, but the proline residue seems to mediate the discrimination between the binding partners. Recently, we showed how multiple adaptin-binding motifs provide an avidity effect where the overall apparent affinities for AP2 complexes will depend on the type, number, and spacing of binding motifs as well as the clustering of available appendage domains (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar, 8Schmid E.M. Ford M.G. Burtey A. Praefcke G.J. Peak-Chew S.Y. Mills I.G. Benmerah A. McMahon H.T. PLoS Biol. 2006; 4: e262Crossref PubMed Scopus (193) Google Scholar). The unclustered cytoplasmic AP2 complex has single α- and β2-appendage domains, whereas in the coated pit there will be many such domains from clustered AP2 complexes. However, when AP2 complexes are bound to polymerized clathrin, the β2-adaptin appendage domain is predicted to be largely occupied by clathrin, and steric hindrance is thought to exclude most accessory proteins. AP2 complexes at the edge of coated pits, where there is a lower concentration of clathrin, are predicted to still be free to bind the full complement of accessory proteins. The complement of accessory proteins bound to AP2 complexes in the cytosol is again proposed to be different (8Schmid E.M. Ford M.G. Burtey A. Praefcke G.J. Peak-Chew S.Y. Mills I.G. Benmerah A. McMahon H.T. PLoS Biol. 2006; 4: e262Crossref PubMed Scopus (193) Google Scholar). Thus, Eps15 has been shown to be restricted to the edges of a nascent coated pit as its interaction with AP2 adaptors is displaced by clathrin (24Edeling M.A. Mishra S.K. Keyel P.A. Steinhauser A.L. Collins B.M. Roth R. Heuser J.E. Owen D.J. Traub L.M. Dev. Cell. 2006; 10: 329-342Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 25Tebar F. Sorkina T. Sorkin A. Ericsson M. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). A similar scenario applies for amphiphysin1, which contains an FXDXF, a DPF, and a DLW motif, the latter two of which overlap with two clathrin-binding motifs (Fig. 1A). As a result, amphiphysin would also be primarily localized to the edges of the invaginating pit, ending up at the neck of the nascent vesicle. This would be a convenient way for amphiphysin to ensure that its C-terminal SH3 domain delivers dynamin to the neck of a coated pit, whereas the N-terminal BAR domain of amphiphysin binds to PtdIns(4,5)P2 and generates vesicle curvature in the membrane (26Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303: 495-499Crossref PubMed Scopus (1354) Google Scholar). The BAR domain is also responsible for the dimerization of amphiphysin, which increases the efficiency of binding to the proline-rich domain in dynamin. Mammals have two isoforms of amphiphysin (27Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (209) Google Scholar), amphiphysin1 and -2 (Amph1 and -2). There is low sequence conservation of the domain in which these motifs occur. However, only the FEDNF motif is well conserved (as an FEDAF in Amph2), whereas the DPF motif is EPL in Amph2. The muscle form of Amph2 (bridging integrator-1/Bin1) and amphiphysin in Drosophila have no DXF motifs in this region. In muscle, the protein is associated with T-tubule formation and not with clathrin/adaptor endocytosis. Accordingly, in Drosophila deletion of the protein gives a muscle weakness phenotype and a defect in T-tubule formation (28Razzaq A. Robinson I.M. McMahon H.T. Skepper J.N. Su Y. Zelhof A.C. Jackson A.P. Gay N.J. O'Kane C.J. Genes Dev. 2001; 15: 2967-2979Crossref PubMed Scopus (187) Google Scholar, 29Lee E. Marcucci M. Daniell L. Pypaert M. Weisz O.A. Ochoa G.C. Farsad K. Wenk M.R. De Camilli P. Science. 2002; 297: 1193-1196Crossref PubMed Scopus (326) Google Scholar). Thus, the presence of a clathrin/adaptor-binding domain targets amphiphysin for function in clathrin-mediated endocytosis. The selection of amphiphysin1 to study the individual contributions of different binding motifs for α-adaptin offers several advantages over other accessory proteins such as epsin1, Eps15, or AP180. First, amphiphysin is highly specific for the α-adaptin appendage and binds to other adaptins weakly (17Owen D.J. Vallis Y. Pearse B.M. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar), thereby avoiding avidity effects from interactions with other AP components. Second, amphiphysin contains single copies of the possible adaptin-binding motifs, which limits interference by avidity effects and makes mutagenesis straightforward. These motifs are separated by 20-30 residues, which should be distant enough to avoid steric hindrance. Finally, despite the low number of motifs, the binding has a high affinity and amphiphysin is a major AP2-binding partner with an essential biological function. Using a combination of thermodynamic, biochemical, and structural observations, we show that the FXDXF motif is the main determinant for the high affinity binding to the α-adaptin appendage. Moreover, we define the optimal FXDXF motif and use these data to explain the observed binding characteristics of other FXDXF- and DX(F/W)-containing accessory proteins. Constructs and Protein Expression–The α-adaptin appendage domain (residues 701-938) and the appendage-plus hinge domain (residues 653-938), the human β2-adaptin appendage domain (residues 701-937), rat amphiphysin1AB (Amph1AB; residues 1-378), and rat amphiphysin2AB (Amph2AB; residues 1-422) were expressed as N-terminal glutathione S-transferase (GST) fusion proteins (in pGex4T2) in BL21 cells following overnight isopropyl 1-thio-β-d-galactopyranoside induction at 22 °C. All GST fusion proteins were purified from bacterial extracts by incubation with glutathione-Sepharose beads, followed by extensive washing with 20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm DTT. The fusion proteins were cleaved by incubation for 2 h with thrombin and further purified by passage over a Q-Sepharose column. For isothermal titration calorimetry (ITC) experiments, the protein was additionally passed down a Superdex 75 gel filtration column and dialyzed into 100 mm HEPES, pH 7.4, 50 mm NaCl, 2 mm DTT. GST-Amph1AB was not cleaved to prevent degradation of the protein. Myc-tagged proteins (in pCMV-myc) were used for expression in COS-7 fibroblasts. The appendage domain of human β2-adaptin (residues 701-937) was expressed in BL21 cells as an N-terminal His6 fusion protein (in pET-15b) and purified by passage over nickel-nitrilotriacetic acid, followed by Q-Sepharose and gel filtration chromatography. Mutations were generated by PCR using overlapping primers incorporating the base pair changes. Transfections, Antibodies, and Cell Extracts–COS-7 cells were transfected using GeneJuice (Novagen) according to the manufacturer's protocol. Overexpressed Amph1AB was detected using a polyclonal anti-Myc antibody (Cell Signaling, green in merged images). The endogenous AP2 complex was detected using a Sigma monoclonal antibody (red in merged images). Cells were imaged using a Bio-Rad Radiance confocal system. For extracts, two 70-mm dishes of COS cells were scraped in 1 ml of phosphate-buffered saline + 0.1% Triton X-100, or one rat brain was homogenized in 10 ml of phosphate-buffered saline + 0.1% Triton X-100 and cleared by centrifugation. Pull Downs from COS-7 Cell or Rat Brain Extracts with GST Appendages–For interaction experiments, the extracts described above were incubated with 30-50 μg of GST fusion protein on glutathione-Sepharose beads for 1 h at 4 °C, and then the bead-bound proteins were washed four times with 150 mm NaCl, 20 mm HEPES, pH 7.4, 2 mm DTT, protease inhibitors, and 0.1% Triton X-100. Interaction partners were analyzed by SDS-PAGE and Western blotting. All experiments were repeated three to five times. The α- and δ-adaptin and amphiphysin1 were detected using monoclonal antibodies from BD Biosciences, and β1,2- and γ-adaptin were detected with monoclonal antibodies from Sigma. Isothermal Titration Calorimetry–Binding of peptides and proteins to α- and β2-adaptin appendage domains was investigated by isothermal titration calorimetry (30Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2437) Google Scholar) using a VP-ITC (MicroCal Inc.). All experiments were performed in 100 mm HEPES, pH 7.4, 50 mm NaCl, 2 mm DTT at 10 °C unless otherwise stated. The peptides or proteins were injected from a syringe in 40-50 steps up to a 3-4-fold molar excess. The cell contained 1.36 ml of protein solution, and typically the ligand was added in steps of 4-8 μl every 3.5 min. Concentrations were chosen so that the binding partners in the cell were at least 5-fold higher than the estimated dissociation constant, if possible. The ligands in the syringe were again at least 10-fold more concentrated. Titration curves were fitted to the data using ORIGIN (supplied by the manufacturer) yielding the stoichiometry N, the binary equilibrium constant Ka (= Kd-1), and the enthalpy of binding ΔH. The entropy of binding ΔS was calculated from the relationship ΔG =-RT·lnKa and the Gibbs-Helmholtz equation. The values were averaged from two to three titrations. Protein concentrations were determined by measuring the A280. Peptides were purchased at >95% purity from the Institute of Biomolecular Sciences, University of Southampton, UK, and weighed on an analytical balance. Where possible, peptide concentrations were verified by measuring A280 or A257. The resulting errors on the concentrations are estimated to be <10% for the proteins and the peptides. Unless otherwise stated, the values for the stoichiometry N were within this error region around N = 1. Crystallography and Structure Determination–Co-crystals of α-adaptin appendage and the synaptojanin WVXF peptide were grown by hanging-drop vapor diffusion against a reservoir containing conditions centered around 1.2 m ammonium sulfate, 3% isopropyl alcohol, and 0.05 m sodium citrate. Hanging drops were 2 μl and contained 222 μm α-adaptin appendage and 277.5 μm synaptojanin WVXF peptide (sequence NPKG-WVTFEEEE). Crystals were obtained after incubation for ∼1 week at 18 °C. To obtain crystals containing peptides bound to the top side, α-adaptin appendage-WVXF co-crystals were soaked in a solution of mother liquor containing amphyphysin1 FEDNFVP peptide. Crystals were cryo-protected by transfer to Paretone-N (Hampton Research) and were flash-cooled in liquid nitrogen. Data to 1.6 Å were collected at 100 K at Station 9.6 SRS Daresbury, UK. Crystals were monoclinic and belonged to space group C2 (a = 146.6 Å, b = 67.3 Å, c = 39.7 Å, β = 94.53°). Data collection statistics are shown insupplemental Table 1. Reflections were integrated using MOSFLM (31Leslie A.W.G. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. 26. Daresbury Laboratory, Warrington, UK1992Google Scholar) and were scaled using the CCP4 suite of crystallographic software (32Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). A difference Fourier, calculated using our model of the α-appendage bound to the WVXF peptide, 8M. G. J. Ford and G. J. K. Praefcke, unpublished data. revealed beautiful density for the amphiphysin peptide. The model was completed using COOT (33Emsley P. Cowtan K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23389) Google Scholar) and O (34Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) and was refined using REFMAC5 (35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar). The validated coordinates and structure factors for the crystal structure containing the synaptojanin WVXF and the amphyphysin1 FEDNFVP have been deposited in the Protein Data Bank (36Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27555) Google Scholar) (PDB code 2vj0). Figures were generated using Aesop, 9M. Noble, personal communication. and the peptide interaction map was generated using the output from LIGPLOT (37Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4368) Google Scholar) as a starting point. The region in amphiphysin1 necessary for binding to the AP2 complex has been mapped to residues 322-340, and binding is enhanced if the region is extended to 322-363 (38Slepnev V.I. Ochoa G.C. Butler M.H. De Camilli P. J. Biol. Chem. 2000; 275: 17583-17589Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This includes the FEDNF328 and the DPF359 motifs (Fig. 1A). A further extension to include the PWXXW had no additional effect on binding. Using isothermal titration calorimetry, we found that a construct from residues 1-378, which forms a dimer because of its N-terminal BAR domain (26Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. Science. 2004; 303: 495-499Crossref PubMed Scopus (1354) Google Scholar), binds to the α-adaptin appendage with an affinity of 1.6 μm but does not interact with the β2-adaptin appendage (Fig. 1B). This affinity is very similar to the one measured for the 12-mer DNF-peptide INFFEDNFVPEI (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar). In addition, the stoichiometry of the interaction between the α-adaptin appendage and the amphiphysin protein, as well as the DNF 12-mer, is 1:1. A longer construct, residues 1-390, comprising the FXDXF, DPF, and the PWXXW motifs, had a similar affinity, although a contribution of a second very weak binding site becomes visible (data not shown). Thus, the FEDNF in amphiphysin1 is the major determinant for the binding to the α-adaptin appendage. Adaptor Binding by Amphiphysin via FXDNF and DPF Motifs–We extended these observations by mutagenesis of both motifs sequentially and simultaneously and show that AP2 binding to the DNF motif at residue 326 is stronger than binding to the DPF at residue 357 (Fig. 2A). Mutations of both DNF and DPF motifs to SGA are even more effective than the single mutants in disrupting binding. In pulldown experiments from brain and COS-7 cell extracts, α- and β-adaptins follow the same pattern, because they are part of the same complex (AP2). We already know from Fig. 1 that amphiphysin is specific for α-adaptin. There is also no interaction of γ- and δ-adaptins with amphiphysin, showing that AP1 and AP3 complexes in COS-7 extracts do not bind to amphiphysin (Fig. 2A). From this it is clear that both motifs contribute to AP2 binding, but there is a clear difference in affinity for the α-adaptin appendage. In Amph2 the FEDAF motif is the major binding sequence for AP2 adaptors (Fig. 2C). Given the strong effect of mutagenesis of the FXDXF motifs in amphiphysin1 and -2, we tested how well other residues might substitute (Fig. 2, B and C). The initial surprise was that FXDPF does not substitute for FXDNF but that FXDPW does. The structural basis for this is not clear, but different peptides can bind in different conformations. The FEDNF peptide from amphiphysin and the FKDSF from synaptojanin bind in an extended conformation where the first F in this binds into the pocket formed by Phe-836, Phe-837, and Trp-840 (7Praefcke G.J. Ford M.G. Schmid E.M. Olesen L.E. Gallop J.L. Peak-Chew S.Y. Vallis Y. Babu M.M. Mills I.G. McMahon H.T. EMBO J. 2004; 23: 4371-4383Crossref PubMed Scopus (147) Google Scholar, 18Brett T.J. Traub L.M. Fremont D.H. Structure (Lond.). 2002; 10: 797-809Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). This cannot be the case for most DX(F/W) peptides that do not have an equivalent Phe residue, and the proline residue in the DP(F/W) motifs forces the peptide into a loop structure. Binding is also possible when the Asn residue in the FEDNF motif of amphiphysin1 is replaced by Ser, Ala, Asp, or Ile, whereas Gly and Leu abolish the interaction. For amphiphysin2, we looked at a more limited set of substitutions (Fig. 2C), but again the change of FXDAF to FXDPF weakens the interaction. The FXDXFis the major binding motif of amphiphysins; however, we should be very cautious in extending this observation to other proteins that have other nonconserve" @default.
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• W1969667037 title "Solitary and Repetitive Binding Motifs for the AP2 Complex α-Appendage in Amphiphysin and Other Accessory Proteins" @default.
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