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• W2160997906 abstract "AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1. AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1. Crystal structure of the AP-1 clathrin adaptor complex bound to Arf1 Arf1 promotes the open conformation and cargo binding by AP-1 AP-1 is recruited via canonical Arf1 contacts with β and γ subunits AP-1 is activated by dimerizing via the back side of Arf1 Clathrin-coated vesicles (CCVs) play major roles in the intracellular transport of selected cargo molecules from the plasma membrane, trans-Golgi network (TGN), and endosomes (Brodsky et al., 2001Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Biological basket weaving: formation and function of clathrin-coated vesicles.Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar; Kirchhausen, 2000Kirchhausen T. Clathrin.Annu. Rev. Biochem. 2000; 69: 699-727Crossref PubMed Scopus (500) Google Scholar). CCV formation starts with the recruitment of adaptor proteins (APs) from the cytosol to the target membranes. The membrane-bound APs interact with sorting signals contained within the cytosolic tails of transmembrane cargo proteins while also inducing the polymerization of clathrin into a polyhedral, lattice-like scaffold. Clathrin-coated membranes curve, eventually leading to the budding of CCVs that contain specific sets of cargo molecules. The main clathrin APs are two homologous, heterotetrameric complexes named AP-1 (γ-β1-μ1-σ1) and AP-2 (α-β2-μ2-σ2) (subunit composition in parentheses), which function at the TGN and endosomes (AP-1) and plasma membrane (AP-2) (Owen et al., 2004Owen D.J. Collins B.M. Evans P.R. Adaptors for clathrin coats: structure and function.Annu. Rev. Cell Dev. Biol. 2004; 20: 153-191Crossref PubMed Scopus (357) Google Scholar; Robinson, 2004Robinson M.S. Adaptable adaptors for coated vesicles.Trends Cell Biol. 2004; 14: 167-174Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Both complexes are structured as a “core” domain, comprising the N-terminal “trunk” portions of γ/α and β1/β2 plus the whole μ1/μ2 and σ1/σ2 subunits and two “appendage” domains, corresponding to the C-terminal portions of γ/α and β1/β2, which are connected to the core by two long, largely unstructured “hinge” sequences. The core domain mediates recruitment to membranes and recognition of sorting signals while the hinge-ear domains interact with clathrin and various accessory proteins. Both AP-1 and AP-2 recognize at least two types of sorting signal: tyrosine-based YXXØ-type signals through binding to the μ1/μ2 subunits (Boll et al., 1996Boll W. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes.EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (236) Google Scholar; Ohno et al., 1995Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Interaction of tyrosine-based sorting signals with clathrin-associated proteins.Science. 1995; 269: 1872-1875Crossref PubMed Scopus (823) Google Scholar, Ohno et al., 1996Ohno H. Fournier M.C. Poy G. Bonifacino J.S. Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains.J. Biol. Chem. 1996; 271: 29009-29015Crossref PubMed Scopus (250) Google Scholar; Owen and Evans, 1998Owen D.J. Evans P.R. A structural explanation for the recognition of tyrosine-based endocytotic signals.Science. 1998; 282: 1327-1332Crossref PubMed Scopus (453) Google Scholar) and dileucine-based [DE]XXXL[LI]-type signals through binding to a site at the interface of the γ-σ1 and α-σ2 subunits (amino acids in single letter code; X is any amino acid, and Ø is a bulky hydrophobic amino acid) (Chaudhuri et al., 2007Chaudhuri R. Lindwasser O.W. Smith W.J. Hurley J.H. Bonifacino J.S. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor.J. Virol. 2007; 81: 3877-3890Crossref PubMed Scopus (168) Google Scholar; Doray et al., 2007Doray B. Lee I. Knisely J. Bu G.J. Kornfeld S. The gamma/sigma1 and alpha/sigma2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site.Mol. Biol. Cell. 2007; 18: 1887-1896Crossref PubMed Scopus (131) Google Scholar; Janvier et al., 2003Janvier K. Kato Y. Boehm M. Rose J.R. Martina J.A. Kim B.Y. Venkatesan S. Bonifacino J.S. Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes.J. Cell Biol. 2003; 163: 1281-1290Crossref PubMed Scopus (187) Google Scholar; Kelly et al., 2008Kelly B.T. McCoy A.J. Späte K. Miller S.E. Evans P.R. Höning S. Owen D.J. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex.Nature. 2008; 456: 976-979Crossref PubMed Scopus (233) Google Scholar; Mattera et al., 2011Mattera R. Boehm M. Chaudhuri R. Prabhu Y. Bonifacino J.S. Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants.J. Biol. Chem. 2011; 286: 2022-2030Crossref PubMed Scopus (79) Google Scholar). The mechanisms of signal recognition and membrane recruitment have been worked out in greatest detail for AP-2. Biochemical and X-ray crystallographic analyses have shown that the AP-2 core occurs in two distinct conformations: a cytosolic, “locked” conformation where binding sites for YXXØ and [DE]XXXL[LI] signals are occluded by portions of β2 (Collins et al., 2002Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Molecular architecture and functional model of the endocytic AP2 complex.Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) and a membrane-bound, “open” conformation where these binding sites are exposed (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The AP-2 core also has four clusters of basic residues (one cluster each on α and β2, and two on μ2) that serve as binding sites for the headgroups of membrane phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] (Collins et al., 2002Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Molecular architecture and functional model of the endocytic AP2 complex.Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar; Gaidarov et al., 1996Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway.J. Biol. Chem. 1996; 271: 20922-20929Crossref PubMed Scopus (147) Google Scholar; Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar; Rohde et al., 2002Rohde G. Wenzel D. Haucke V. A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis.J. Cell Biol. 2002; 158: 209-214Crossref PubMed Scopus (134) Google Scholar). In the locked conformation, the μ2 C-terminal domain responsible for binding to the YXXØ signal is sequestered in a bowl formed by the trunk domains of the α and β2 subunits. In the open conformation, the two signal-binding sites and four PI(4,5)P2-binding sites become coplanar, enabling simultaneous interactions with cargo proteins and PI(4,5)P2 and thus stabilizing the open conformation of the core (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The enrichment of PI(4,5)P2 at the plasma membrane (Di Paolo and De Camilli, 2006Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2070) Google Scholar) ensures that AP-2 is specifically recruited to this compartment. The structural bases for AP-1 signal recognition and membrane recruitment are less well understood. The AP-1 core also occurs in a locked conformation similar to that of the AP-2 core, as shown by X-ray crystallography (Heldwein et al., 2004Heldwein E.E. Macia E. Wang J. Yin H.L. Kirchhausen T. Harrison S.C. Crystal structure of the clathrin adaptor protein 1 core.Proc. Natl. Acad. Sci. USA. 2004; 101: 14108-14113Crossref PubMed Scopus (96) Google Scholar). The existence of an open conformation of the AP-1 core has not been demonstrated by structural methods but is supported by other lines of evidence. First, the residues that bind YXXØ and [DE]XXX[LI] signals in AP-2 (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar; Kelly et al., 2008Kelly B.T. McCoy A.J. Späte K. Miller S.E. Evans P.R. Höning S. Owen D.J. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex.Nature. 2008; 456: 976-979Crossref PubMed Scopus (233) Google Scholar; Owen and Evans, 1998Owen D.J. Evans P.R. A structural explanation for the recognition of tyrosine-based endocytotic signals.Science. 1998; 282: 1327-1332Crossref PubMed Scopus (453) Google Scholar) are highly conserved in AP-1 (Heldwein et al., 2004Heldwein E.E. Macia E. Wang J. Yin H.L. Kirchhausen T. Harrison S.C. Crystal structure of the clathrin adaptor protein 1 core.Proc. Natl. Acad. Sci. USA. 2004; 101: 14108-14113Crossref PubMed Scopus (96) Google Scholar), and mutation of these residues abrogates binding of both types of signal to AP-1 in yeast two- and three-hybrid assays (Carvajal-Gonzalez et al., 2012Carvajal-Gonzalez J.M. Gravotta D. Mattera R. Diaz F. Perez Bay A. Roman A.C. Schreiner R.P. Thuenauer R. Bonifacino J.S. Rodriguez-Boulan E. Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B.Proc. Natl. Acad. Sci. USA. 2012; 109: 3820-3825Crossref PubMed Scopus (62) Google Scholar; Mattera et al., 2011Mattera R. Boehm M. Chaudhuri R. Prabhu Y. Bonifacino J.S. Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants.J. Biol. Chem. 2011; 286: 2022-2030Crossref PubMed Scopus (79) Google Scholar). Second, binding of one type of signal enhances binding of the other type, probably due to stabilization of an open conformation (Lee et al., 2008aLee I. Doray B. Govero J. Kornfeld S. Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP.J. Cell Biol. 2008; 180: 467-472Crossref PubMed Scopus (37) Google Scholar). Whereas the mechanisms of signal recognition by AP-1 and AP-2 appear quite similar, the determinants of recruitment to their corresponding membranes differ significantly. The AP-1 core has a phosphoinositide-binding site with preference for phosphatidylinositol-4-phosphate [PI(4)P] on its γ subunit, at a location similar to that of the PI(4,5)P2-binding site on AP-2 α (Heldwein et al., 2004Heldwein E.E. Macia E. Wang J. Yin H.L. Kirchhausen T. Harrison S.C. Crystal structure of the clathrin adaptor protein 1 core.Proc. Natl. Acad. Sci. USA. 2004; 101: 14108-14113Crossref PubMed Scopus (96) Google Scholar; Wang et al., 2003Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi.Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). PI(4)P is enriched within domains of the TGN and endosomes (Di Paolo and De Camilli, 2006Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2070) Google Scholar), consistent with the association of AP-1 to these compartments. In contrast to the case of AP-2, however, phosphoinositides alone are insufficient to recruit AP-1 to its sites of action. Instead, the key determinant of AP-1 targeting to the TGN and endosomes is its interaction with members of the ADP ribosylation factor (Arf) family of small GTPases, particularly Arf1 (Seaman et al., 1996Seaman M.N.J. Sowerby P.J. Robinson M.S. Cytosolic and membrane-associated proteins involved in the recruitment of AP-1 adaptors onto the trans-Golgi network.J. Biol. Chem. 1996; 271: 25446-25451Crossref PubMed Scopus (64) Google Scholar; Stamnes and Rothman, 1993Stamnes M.A. Rothman J.E. The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein.Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (339) Google Scholar; Traub et al., 1993Traub L.M. Ostrom J.A. Kornfeld S. Biochemical dissection of AP-1 recruitment onto Golgi membranes.J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Arf1 cooperates with cargo and phosphoinositides such that AP-1 binding to all of these components is thought to be necessary for targeting under normal conditions (Crottet et al., 2002Crottet P. Meyer D.M. Rohrer J. Spiess M. ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes.Mol. Biol. Cell. 2002; 13: 3672-3682Crossref PubMed Scopus (47) Google Scholar; Le Borgne et al., 1996Le Borgne R. Griffiths G. Hoflack B. Mannose 6-phosphate receptors and ADP-ribosylation factors cooperate for high affinity interaction of the AP-1 Golgi assembly proteins with membranes.J. Biol. Chem. 1996; 271: 2162-2170Crossref PubMed Scopus (101) Google Scholar; Lee et al., 2008aLee I. Doray B. Govero J. Kornfeld S. Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP.J. Cell Biol. 2008; 180: 467-472Crossref PubMed Scopus (37) Google Scholar), although enrichment of cargo signals to high levels can override this requirement (Lee et al., 2008bLee I. Drake M.T. Traub L.M. Kornfeld S. Cargo-sorting signals promote polymerization of adaptor protein-1 in an Arf-1.GTP-independent manner.Arch. Biochem. Biophys. 2008; 479: 63-68Crossref PubMed Scopus (7) Google Scholar). Arfs cycle between a GDP-bound, inactive cytosolic form and a GTP-bound, active membrane-tethered form (Donaldson and Jackson, 2011Donaldson J.G. Jackson C.L. ARF family G proteins and their regulators: roles in membrane transport, development and disease.Nat. Rev. Mol. Cell Biol. 2011; 12: 362-375Crossref PubMed Scopus (594) Google Scholar). Conversion to the GTP-bound form requires a guanine nucleotide exchange factor (GEF), whereas conversion to the GDP-bound form is catalyzed by a GTPase activating protein (GAP). Loading with GTP causes Arfs to undergo a conformational change, exposing a myristoylated N-terminal amphipathic helix that inserts into the membrane while reconfiguring its switch I-II and interswitch regions to allow the binding of effector proteins (Donaldson and Jackson, 2011Donaldson J.G. Jackson C.L. ARF family G proteins and their regulators: roles in membrane transport, development and disease.Nat. Rev. Mol. Cell Biol. 2011; 12: 362-375Crossref PubMed Scopus (594) Google Scholar). Arf1 has many effectors, including AP-1 and the homologous heterotetrameric complexes AP-3 (Ooi et al., 1998Ooi C.E. Dell’Angelica E.C. Bonifacino J.S. ADP-Ribosylation factor 1 (ARF1) regulates recruitment of the AP-3 adaptor complex to membranes.J. Cell Biol. 1998; 142: 391-402Crossref PubMed Scopus (166) Google Scholar), AP-4 (Boehm et al., 2001Boehm M. Aguilar R.C. Bonifacino J.S. Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs).EMBO J. 2001; 20: 6265-6276Crossref PubMed Scopus (79) Google Scholar), and COPI (F subcomplex) (Serafini et al., 1991Serafini T. Orci L. Amherdt M. Brunner M. Kahn R.A. Rothman J.E. ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.Cell. 1991; 67: 239-253Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Arf1 is not enriched at the plasma membrane and is not thought to interact with AP-2 in cells. Some studies, however, have suggested that the plasma-membrane-associated Arf6 could be involved in recruiting AP-2 (Krauss et al., 2003Krauss M. Kinuta M. Wenk M.R. De Camilli P. Takei K. Haucke V. ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.J. Cell Biol. 2003; 162: 113-124Crossref PubMed Scopus (235) Google Scholar; Montagnac et al., 2011Montagnac G. de Forges H. Smythe E. Gueudry C. Romao M. Salamero J. Chavrier P. Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling.Curr. Biol. 2011; 21: 574-579Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar; Paleotti et al., 2005Paleotti O. Macia E. Luton F. Klein S. Partisani M. Chardin P. Kirchhausen T. Franco M. The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes.J. Biol. Chem. 2005; 280: 21661-21666Crossref PubMed Scopus (88) Google Scholar; Poupart et al., 2007Poupart M.-E. Fessart D. Cotton M. Laporte S.A. Claing A. ARF6 regulates angiotensin II type 1 receptor endocytosis by controlling the recruitment of AP-2 and clathrin.Cell. Signal. 2007; 19: 2370-2378Crossref PubMed Scopus (31) Google Scholar). Thus, the question of how Arf-family GTPases recognize, recruit, and activate AP complexes has broad implications for intracellular traffic. Recently, important insight into Arf1 recognition was obtained from the structure of a truncated γζ subcomplex from COPI (Yu et al., 2012Yu X.C. Breitman M. Goldberg J. A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes.Cell. 2012; 148: 530-542Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The goal of the present study was to take the next step in understanding whether Arf1 regulates not only the localization, but also the conformation of heterotetrameric sorting complexes. To address this question, we solved the crystal structure of the AP-1 core in complex with GTP-bound Arf1. The most important insight is that Arf1-GTP alone, in the absence of cargo or PI(4)P, can unlock AP-1 and drive it into the open conformation. AP-1 contains two binding sites for the canonical switch I and II surface on Arf1, one on each of the two trunk domains. Both of these Arf1-binding sites are required for high-affinity binding in vitro and for subcellular localization to the TGN and endosomes, but only the site on β1 is important for activation. Moreover, a surface on the C-terminal portion (“back side”) of Arf1, distal to switch I and II, was found to be required for full allosteric activation, although it does not contribute to recruitment. Taking together the mutational and biochemical analyses with the dimeric assemblage of AP-1 in the crystal lattice, we deduced a model for the allosteric activation mechanism. Reconstitution of the recruitment of AP-1 to liposomes by Arf1, cargo, and PI(4)P highlights the profound cooperativity between the binding of cargo to AP-1 and Arf1. The core of the AP-1 adaptor complex was reconstituted by coexpressing the trunk domains of the murine γ1 (residues 1–595) and human β1 (residues 1–584) subunits with full-length human σ1C and murine μ1A subunits in E. coli using a single polycistronic expression plasmid. Human Arf1 bearing the GTPase mutation Q71L and the N-terminal truncation Δ1-16 (Arf1Δ1-16) was loaded with GTP and mixed at a 4:1 excess of Arf1 relative to AP-1. Crystals were obtained that diffracted to 7.0 Å resolution. The crystal structure of the AP-1:Arf1-GTP complex (Figure 1A) was determined by the molecular replacement method. Because it was not known a priori whether the crystallized AP-1 core would be in one of the expected locked or open conformations, or in some novel conformation, test searches were run using all of the available crystal structures of AP-1 and AP-2 core complexes. A solution was obtained using as a search model the core of AP-2 in the open conformation (PDB: 2XA7) (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). At this stage, clear (Fo−Fc)αcalc difference electron density was visible for the entire Arf1Δ1-16 molecule (Figure 1B). The 1.6 Å structure of the GTP-bound form of murine Arf1Δ1-17 (Shiba et al., 2003Shiba T. Kawasaki M. Takatsu H. Nogi T. Matsugaki N. Igarashi N. Suzuki M. Kato R. Nakayama K. Wakatsuki S. Molecular mechanism of membrane recruitment of GGA by ARF in lysosomal protein transport.Nat. Struct. Biol. 2003; 10: 386-393Crossref PubMed Scopus (103) Google Scholar) (PDB: 1O3Y) was used as a search model to position the molecule in the unit cell. The clarity of this unbiased difference map, in spite of its low resolution, persuaded us to refine the structure and characterize its functional implications. It is not possible to visualize side chains at this resolution. However, given the availability of well-refined starting models for substructures, contemporary refinement methodology makes it possible to accurately analyze the overall conformation of large protein complexes and the nature of their interfaces from diffraction data at as low as 7 Å resolution (Brunger et al., 2012Brunger A.T. Adams P.D. Fromme P. Fromme R. Levitt M. Schröder G.F. Improving the accuracy of macromolecular structure refinement at 7 Å resolution.Structure. 2012; 20: 957-966Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Refinement of this structure was facilitated by the finding that the Arf1-bound conformation of AP-1 is nearly identical to the open conformation of AP-2, which was refined at 3.1 Å resolution (Jackson et al., 2010Jackson L.P. Kelly B.T. McCoy A.J. Gaffry T. James L.C. Collins B.M. Höning S. Evans P.R. Owen D.J. A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.Cell. 2010; 141: 1220-1229Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The main chain of the AP-1 structure was tethered to a model based on the open conformation of AP-2 using the deformable elastic network (DEN) methodology (Schröder et al., 2010Schröder G.F. Levitt M. Brunger A.T. Super-resolution biomolecular crystallography with low-resolution data.Nature. 2010; 464: 1218-1222Crossref PubMed Scopus (243) Google Scholar). A starting model of AP-1 in this conformation was generated by superimposing the 4.0 Å resolution coordinates of the AP-1 core complex (PDB: 1W63) (Heldwein et al., 2004Heldwein E.E. Macia E. Wang J. Yin H.L. Kirchhausen T. Harrison S.C. Crystal structure of the clathrin adaptor protein 1 core.Proc. Natl. Acad. Sci. USA. 2004; 101: 14108-14113Crossref PubMed Scopus (96) Google Scholar) onto the open conformation of AP-2 on a domain-by-domain basis. The trunk domains of β1 and γ were broken into three fragments for the superposition, and μ1 was broken into its N- and C-terminal domains. All of these domains had excellent fits with the sole exception of the μ1 C-terminal domain. Therefore, the μ1 C-terminal domain model was derived by replacing the side chains of μ2 in the TGN38-bound structure with their cognates from μ1. Even though the side chains were not visualized, they were included in the refinement in order to account for their contribution to X-ray scattering. Side-chain conformations were allowed to relax in order to accommodate sequence differences with respect to the parent models used for molecular replacement and to avoid steric collisions at Arf1 binding and lattice interfaces. The resulting structure (Figure 1A) had a free R-factor of 0.25 and excellent stereochemistry (Table S1, available online). Moreover, as described below, the structural interfaces underwent extensive validation on the Arf1 and AP-1 sides, both in solution and in cells. By analogy to AP-2, it was anticipated that AP-1 would be activated through a conformational change and exposure of the YXXØ- and [DE]XXXL[LI]-binding sites. Here, we have visualized the active conformation of AP-1 in the presence of Arf1-GTP but in the absence of cargo tails, phosphoinositides, or soluble phosphoinositide analogs. The overall structure is essentially superimposable on that of the YXXØ-bound AP-2 core (Figure 1C), which is the structural paradigm of the active conformation. This observation is consistent with the biochemical evidence that Arf1-GTP is a direct allosteric activator of AP-1. The crystals contain one copy each of the AP-1 core and Arf1 (Figure 1A). Arf1 bridges two copies of the AP-1 core in the crystal lattice, such that Arf1 binds to two sites on AP-1 (Figure 1D). The larger of the two interfaces (∼720 Å2) buries the switch I and II regions of Arf1 against helices α1, α3, and α5 of the β1 subunit (Figures 2A and 2B ). The β1 contact is centered on Gln59, Ile85, and Asn89. Arf1 contacts include Ile46, Ile49, Gly50, Phe51, Asn52, and Val53 of switch I; Trp66, Lys73, Ile74, Leu77, His80, Tyr81, and Gln83 of switch II; and Tyr35 of α1 (Figures 2A and 2B). Switch I and II are the regions of Arf1 that change conformation upon GTP binding (Goldberg, 1998Goldberg J. Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching.Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). The GDP-bound conformation of Arf1 (Amor et al., 1994Amor J.C. Harrison D.H. Kahn R.A. Ringe D. Structure of the human ADP-ribosylation factor 1 complexed with GDP.Nature. 1994; 372: 704-708Crossref PubMed Scopus (252) Google Scholar) is not compatible with the β1 structure because of an extensive clash between switch I and β1-α5 (Figure S1). The involvement of the switch regions in AP-1 binding has been noted (Liang et al., 1997Liang J.O. Sung T.C. Morris A.J. Frohman M.A. Kornfeld S. Different domains of mammalian ADP-ribosylation factor 1 mediate interaction with selected target proteins.J. Biol. Chem. 1997; 272: 33001-33008Crossref PubMed Scopus (37) Google Scholar) and is consistent with the GTP requirement for membrane recruitment of AP-1 and its inhibition by the Arf1 GEF inhibitor brefeldin A (Stamnes and Rothman, 1993Stamnes M.A. Rothman J.E. The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein.Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (339) Google Scholar; Traub et al., 1993Traub L.M. Ostrom J.A. Kornfeld S. Biochemical dissection of AP-1 recruitment onto Golgi membranes.J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). The β1-binding site is in accord with an Arf-1-binding site recently predicted to occur on the α4 and α6 helices of the β-COP subunit of COPI (Yu et al., 2012Yu X.C. Breitman M. Goldberg J. A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes.Cell. 2012; 148: 530-542Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), which correspond to α3 and α5 helices of β1. The direct interaction of the β1 subu" @default.
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• W2160997906 title "Structural Basis for Recruitment and Activation of the AP-1 Clathrin Adaptor Complex by Arf1" @default.
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