Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly
2009; Springer Nature; Volume: 29; Issue: 3 Linguagem: Inglês
10.1038/emboj.2009.383
ISSN1460-2075
AutoresYi Xing, Till Böcking, Matthias Wolf, Nikolaus Grigorieff, Tomas Kirchhausen, Stephen C. Harrison,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoArticle24 December 2009Open Access Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly Yi Xing Yi Xing Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Search for more papers by this author Till Böcking Till Böcking Department of Cell Biology, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Matthias Wolf Matthias Wolf Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Search for more papers by this author Nikolaus Grigorieff Nikolaus Grigorieff Rosenstiel Basic Medical Research Center, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA Search for more papers by this author Tomas Kirchhausen Tomas Kirchhausen Department of Cell Biology, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephen C Harrison Corresponding Author Stephen C Harrison Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yi Xing Yi Xing Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Search for more papers by this author Till Böcking Till Böcking Department of Cell Biology, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Matthias Wolf Matthias Wolf Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Search for more papers by this author Nikolaus Grigorieff Nikolaus Grigorieff Rosenstiel Basic Medical Research Center, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA Search for more papers by this author Tomas Kirchhausen Tomas Kirchhausen Department of Cell Biology, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephen C Harrison Corresponding Author Stephen C Harrison Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Yi Xing1, Till Böcking2, Matthias Wolf1, Nikolaus Grigorieff3, Tomas Kirchhausen2 and Stephen C Harrison 1,4 1Department of Biological Chemistry and Molecular Pharmacology, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA, USA 2Department of Cell Biology, Program in Cellular and Molecular Medicine and Immune Disease Institute, Children's Hospital, Harvard Medical School, Boston, MA, USA 3Rosenstiel Basic Medical Research Center, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA 4Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 432 5607; Fax: +1 617 432 5600; E-mail: [email protected] The EMBO Journal (2010)29:655-665https://doi.org/10.1038/emboj.2009.383 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly. Introduction Clathrin-coated vesicles transport cargo molecules, such as receptor-bound transferrin or LDL, from the plasma membrane to endosomes. Clathrin coats assemble as invaginating ‘pits’ and dissociate after the enclosed vesicle has pinched off from the parent membrane (Roth and Porter, 1964; Anderson et al, 1977; Kirchhausen, 2000; Brett and Traub, 2006). The ATP-dependent chaperone, Hsc70, facilitates uncoating, providing the energy required to drive the clathrin assembly–disassembly cycle (Schmid et al, 1985; Greene and Eisenberg, 1990; Barouch et al, 1994). Like other members of the 70 kDa heat-shock protein family (Hsp70s), Hsc70 is an ATP-driven molecular clamp (Hartl and Hayer-Hartl, 2002). Its N-terminal, nucleotide-binding domain (NBD) couples rounds of nucleotide hydrolysis to stages of opening and closing of its C-terminal, substrate-binding domain. The latter has a groove to receive a hydrophobic peptide and a ‘lid’ to close down over the bound peptide, after hydrolysis of ATP (Zhu et al, 1996). Hsp70s facilitate protein folding, by reducing aggregation and transiently stabilizing exposed, hydrophobic segments, and protein translocation, by preventing back diffusion. But how can a purely local mechanism of action drive a large-scale process like the disassembly of a clathrin coat? Clathrin coats are lattices formed by the interdigitation of trimeric assembly units (triskelions), which have extended legs radiating out from a three-fold hub (Figure 1) (Ungewickell and Branton, 1981; Smith et al, 1998; Musacchio et al, 1999). The packing of individual triskelions is sufficiently flexible that both pentagonal and hexagonal (and occasionally heptagonal) rings can form (Cheng et al, 2007); 12 (or 12 plus the number of heptagonal facets) pentagons generate a closed structure. The symmetrical, D6-barrel lattice shown in Figure 1 can be prepared in reasonably high yield (with respect to other lattices) when clathrin triskelions self-assemble together with the endocytic adaptor, AP-2, under defined conditions in vitro (Fotin et al, 2004b). The structure of such a D6 barrel has been determined by electron cryomicroscopy (cryoEM) and single-particle analysis (Fotin et al, 2004b), to a resolution (about 8 Å) sufficient to place α-carbons of most residues, using as guides high-resolution X-ray crystallographic structures of two different fragments (Ter Haar et al, 1998; Ybe et al, 1999). Each triskelion leg comprises an elongated heavy chain (1675 residues), extending from the globular ‘terminal domain’ at the N-terminus to the hub at the C-terminus, and a light chain. Except for the terminal domain and for about 75 residues at the C-terminus, the entire heavy chain consists of ∼40-residues, α-helical zig-zags, in eight approximate repeats of five zig-zags each. The compliance of the zig-zags allows a leg to adapt to variable curvature at different positions in the coat. The only well-ordered part of the light chain is a 71-residue α-helix, which interacts with a portion of the heavy chain relatively close to the hub. Figure 1.Components of the clathrin uncoating process. (A) Domain organization of Hsc70 (top), auxilin (middle) and clathrin heavy chain (bottom). Residue numbers for domain or regional boundaries are shown below the bars. (B) A clathrin triskelion (left) and its packing within the lattice of a coat (right). The various regions of the heavy chain are labelled; the ordered, 71-residue α-helical segment of the light chain is also shown. Three symmetry-distinct vertices are colour-coded, yellow, blue (the hub of the blue triskelion) and green. (C) Side view of the triskelion (left), illustrating the pucker at the apex, and a close-up of the hub region, including the helical tripod and the QLMLT sequence near the C-terminus. Download figure Download PowerPoint In the lattice, each triskelion leg (heavy chain) extends along three edges. As illustrated in Figure 1, the terminal domain, which projects inwards, connects into the first of the zig-zag repeats. The various zig-zag-repeat segments (linker, ankle, distal leg, knee, proximal leg) have acquired names mostly related to the meaning of ‘triskelion’ as ‘three-legged’. The linker runs along part of an edge. The ankle crosses with two others beneath a vertex. The distal leg spans an edge at an intermediate radius, interacting closely with the proximal leg of another triskelion just ‘above’ it. The knee bends gently at a vertex to allow the hub of the triskelion centred at that vertex to project inward. The proximal leg spans yet another edge and terminates at the three-fold hub structure, which has an inward projecting helical tripod, terminating in the only disordered segment in the entire heavy chain (residues 1630–1675). The C-terminus of the heavy chain thus faces terminal domains of three triskelions, each centred three vertices removed from the hub in question. For so elaborately interdigitated a structure, the molecular contacts are relatively modest. The most extensive interface is the one between distal and proximal legs, mentioned in the preceding paragraph. At neutral pH, assembly requires the additional stability provided by interaction with clathrin adaptors or other accessory proteins and by the tendency of many of these proteins to aggregate, thus nucleating a relatively small structure like the D6 barrel (Vigers et al, 1986; Shih et al, 1995). At pH <6.2, assembly of triskelions into ‘cages’ is spontaneous, but the distribution of sizes is broader. Like all Hsp70 family members, Hsc70 requires a so-called J-domain containing protein to recruit it to a specific substrate (Hartl and Hayer-Hartl, 2002). The clathrin-linked J-domain protein is auxilin, a multi-domain protein that includes, in addition to C-terminal clathrin-binding and J-domain regions, a region with homology to the phosphoinositide phosphatase, PTEN (Ahle and Ungewickell, 1990; Ungewickell et al, 1995, 1997; Haynie and Ponting, 1996; Barouch et al, 1997). The timing of auxilin recruitment to a coated vesicle, immediately after budding, appears to determine its prompt uncoating (Lee et al, 2006; Massol et al, 2006). In vitro, a C-terminal fragment (residues 547–910), which includes the clathrin-binding and J-domain functions, is sufficient for Hsc70- and ATP-dependent uncoating (Holstein et al, 1996). Also required for uncoating in vitro is the C-terminal segment of the heavy chain (Rapoport et al, 2008), which projects inward from the helical tripod within a funnel-like cavity defined by the three heavy-chain ankles that cross at that vertex (Figure 1B and C) (Fotin et al, 2004b). It contains a sequence (QLMLT, residues 1638–1642 in mammalian clathrin) that corresponds closely to the consensus sequence for optimal binding to the substrate groove in Hsc70 (Gragerov et al, 1994). Deletion or mutation of this short segment, or moving it closer to the triskelion hub, does not interfere with assembly, but it renders the assembled coats resistant to Hsc70, auxilin and ATP-dependent dissociation (Rapoport et al, 2008). Binding of auxilin (547–910) to in vitro-assembled, D6-barrel coats saturates at one auxilin fragment per heavy chain (Fotin et al, 2004a). A cryoEM reconstruction has shown that each terminal domain binds an auxilin fragment, which also makes contacts with two other heavy chains in the lattice (Fotin et al, 2004a). The contact surface can explain the reported competition of auxilin with ‘clathrin-box’ peptides that bind the terminal domain (Smith et al, 2004). This location for auxilin is appropriate for recruiting Hsc70 to the vicinity of the C-terminal peptide, its presumptive local substrate. An additional consequence of adding auxilin (547–910) is a change in the overall axial ratio of the barrel-like coat (Fotin et al, 2004a). Thus, even addition of auxilin locks in a global perturbation in the clathrin lattice. We report in this paper the structure of a D6 clathrin barrel bound with Hsc70 recruited by auxilin (547–910), determined by cryoEM and single-particle analysis at 11 Å resolution. The Hsc70 associates with the C-terminal segment, as anticipated, with a stoichiometry of about one per three-fold vertex, giving rise to a globular density feature. ATP hydrolysis must take place to achieve strong Hsc70 binding, consistent with the ATPase cycle described above. Distortion of the clathrin lattice, even beyond the perturbation induced by auxilin (547–910), suggests that when Hsc70 splits ATP and clamps firmly onto the heavy-chain C-terminal segment, it locks in place a transient fluctuation to a locally strained configuration and that introduction of a critical number of such distortions favours disassembly. We propose that we have trapped an early uncoating intermediate, prevented by reduced pH from progressing toward dissociation. Results Preparation of auxilin-bearing clathrin coats with specifically bound Hsc70 Hsc70 is relatively promiscuous in its binding propensity, and at high enough concentrations it associates extensively but non-specifically with clathrin coats. We, therefore, sought conditions under which we could obtain restricted, auxilin-dependent association of Hsc70. We screened for tight binding of Hsc70 (1–554):ATP with in vitro assembled clathrin/AP-2 coats bearing auxilin (547–910), prepared as described earlier (Fotin et al, 2004a). Hsc70 (1–554) is a C-terminally truncated form with diminished tendency to aggregate; it retains ATP- and auxilin-dependent uncoating activity (Jiang et al, 1997, 2005; Ungewickell et al, 1997). Auxilin (547–910) is a fragment sufficient to recruit Hsc70 and to stimulate uncoating; it encompasses the clathrin-binding and J-domains (Holstein et al, 1996) (Figure 1A). As a control for promiscuous, auxilin-independent binding, we used Hsc70 (1–554):ADP, which does not interact with auxilin (Holstein et al, 1996). We observed auxilin (547–910)-dependent binding only in the presence of ATP; non-hydrolysable ATP analogs (AMPPNP, AMPPCP, ATP-γS, ADP-AlF4, ADP-BeF3, ADP-vanadate) gave no increment over background binding. To prevent ATP-stimulated uncoating, we stabilized the coats by carrying out the incubation on ice at pH 6.0. We could saturate the coats by adding excess Hsc70 in a molar ratio to clathrin heavy chain of about 10:1. From Coomassie-blue-stained band intensities from SDS–PAGE, we estimated that at saturation, the Hsc70 bound in excess over the auxilin-independent background was ∼0.5 moles Hsc70 per mole clathrin heavy chain, or between one and two Hsc70 molecules per trimer (Figure 2A). Figure 2.Tight, auxilin-specific binding of Hsc70 depends on ATP hydrolysis. (A) SDS–PAGE of resuspended high-speed pellet from preparation of coats, bound with saturating amounts of auxilin (547–910) and incubated with increasing concentrations of Hsc70:ATP (lanes 1–4) or Hsc70:ADP (lanes 5–8). See Materials and methods for details. (B) Hsc70 associated with coats has hydrolysed ATP. TLC analysis showing 32P-labelled nucleotide in the mixture at the time of Hsc70:ATP addition and after separation by centrifugation into supernatant (free Hsc70 with both free and bound nucleotides) and pellet (Hsc70 and nucleotide bound to coats). Download figure Download PowerPoint The failure of non-hydrolysable ATP analogs to stimulate auxilin-dependent association with coats suggests that ATP hydrolysis is necessary for tight binding. Analysis of the nucleotide composition of the Hsc70-containing coats showed essentially no residual ATP (Figure 2B), under conditions in which substantial quantities of unhydrolysed ATP remained in the solution. We conclude that the preparation we have described yields coats to which auxilin (547–910) has recruited Hsc70:ATP, with subsequent nucleotide hydrolysis. This conclusion is consistent with the known properties of Hsc70 and other Hsp70 homologs: the chaperones associate with J-domain-containing co-chaperones in their ATP-bound form, while subsequent tight attachment to the substrate requires ATP hydrolysis. Electron cryoEM of D6 coats with bound auxilin and Hsc70 We obtained an image reconstruction from about 1500 ‘best’ D6-coat images, selected from the original 14 000-particle stack. Image selection was based on phase residuals at successive stages of refinement (Fotin et al, 2006). The nominal resolution, using a Fourier-shell correlation (FSC) cutoff of 0.143, is 15.2 Å. As the coat has nine copies of the clathrin heavy chain within each D6 asymmetric unit, we could improve the resolution and enhance signal-to-noise by averaging corresponding segments of the triskelion legs, as described (Fotin et al, 2004b, 2006). The FSC-estimated resolution of the non-coat-symmetry (n.c.s.) averaged map is 11.3 Å (Supplementary Figure S1). This estimate is consistent with the appearance of the map, in regions of known molecular structure. Clathrin coats are less rigid and less uniform than icosahedral virus particles or ribosomes, and elimination of particles with high phase residuals selects for minimally distorted coats (Fotin et al, 2004b, 2006). To verify that stringent selection of undistorted particles did not affect the molecular interpretation, we compared the model based on our final map with one of the intermediate maps obtained in the course of refinement—a reconstruction at 21 Å resolution derived from about 7000 particles (Supplementary Figure S2). All the features described and analysed here can be seen in this lower resolution map, which was not subjected to n.c.s. averaging (Supplementary Figure S2). To validate directly that discarded particles with high phase residuals are distorted in some way, we carried out a multi-reference alignment of the complete data set using six classes. The largest class contained about 44% of the particles; the remaining five classes showed clear evidence of distortion or damage (see Materials and methods for details). We fit models for individual segments of the clathrin heavy chain into the n.c.s. (nine-fold) averaged density by visual inspection, followed by computational rigid-body refinement. We used the proximal-leg, distal-leg pair (see next paragraph) as one rigid body, the terminal domain as a second, the ankles as a third, and the C-terminal tripod helix as a fourth. The knee bends variably at each of the nine D6-distinct locations, so a model for that region was fit to connect the appropriately placed, rigid-body refined segments just listed. The model matches well with density features throughout the structure (Figure 3). We also carried out exactly the same density averaging and model fitting procedure with the auxilin (547–910)-bound coat reconstruction (from Fotin et al, 2004a) to facilitate accurate comparison. Figure 3.Image reconstruction of an Hsc70 (1–554):auxilin (547–910):clathrin coat. (A) Outside view (left) and cutaway view (right) of the complete coat. Clathrin is in blue, auxilin (547–910) is in red and Hsc70 (1–554) is in green. The boundaries of clathrin and the auxilin fragment are as in Fotin et al (2004b). The boundary of the Hsc70 was determined by comparing the new reconstruction with the previously published reconstruction of the auxilin complex. (B) Detailed views of the density map in specific regions, to illustrate the helical zig-zag and the fit of the heavy-chain model. Download figure Download PowerPoint It is clear from comparison of the density maps for ‘native’ (Fotin et al, 2004b), auxilin-bound (Fotin et al, 2004a) and Hsc70:auxilin-bound coats (this work) that the association of proximal and distal triskelion legs, which run parallel to each other along an edge and have an extended, radial contact, is essentially invariant, both among n.c.s.-related edges and among the three different states of the coat we have studied. Superposing the proximal segment densities from the three maps (after n.c.s. averaging) results in an excellent match of distal segment densities (Figure 4). This invariance suggests that our Hsc70:auxilin:clathrin coats maintain integrity in the presence of ATP through strengthening of the proximal–distal contact at pH 6. Low pH also favours clathrin assembly in vitro, even in the absence of adaptors or other assembly promoting components, and we propose that it is the invariant proximal–distal interface that determines the stability of these clathrin ‘cages’. Figure 4.Invariance of the proximal–distal contact. (A) The 8 Å resolution map of the D6 coat (Fotin et al, 2004a), with the model of corresponding heavy-chain segments. The view is in a direction tangential to the surface of a coat, with the exterior of the lattice above and the interior below. (B) Corresponding map and model for the Hsc70:auxilin:clathrin complex. (C) Superposition of the two, with the map from the uncomplexed coat in blue (as in A) and the map from the ternary complex in brown (as in B). The two maps were positioned to optimize agreement in the proximal-leg region, and the excellent superposition of the distal-leg maps shows that the interface does not shift when the ligands distort the coat. Download figure Download PowerPoint Auxilin and Hsc70 Density features corresponding to auxilin (547–910) can be identified at three quasi-equivalent positions around each vertex, as described earlier (Fotin et al, 2004a). With reference to the triskelion centred at any particular vertex, each of the three adjacent auxilin fragments contacts the terminal domain from a triskelion centred three vertices away and the ankle region from one centred two vertices away (Figure 5). The C-termini of the reference triskelion project inward, within the triangle of auxilin fragments. The J-domain of auxilin is augmented, at its N-terminus, by two α-helices (Gruschus et al, 2004), and the composite structure (residues 797–910) docks into the maps in an orientation very similar to our earlier fit (Figure 5) (Fotin et al, 2004a). Density for the rest of the fragment (the clathrin-binding region) cannot yet be fit, as there is currently no atomic model for that part of auxilin. Figure 5.Relative positions of auxilin (547–910) and Hsc70 (1–554) in the complex. (A) Overview of the D6 coat, showing in dashed outline the region illustrated in close-up to the right. The lattices at the top and centre are viewed from outside; the lattice at the bottom is cut away at the front, and the indicated hub is viewed from the inside. (B) Close-up view, in surface rendering, of the hub indicated in (A). The triskelion centred at the vertex shown is in orange; triskelions centred at nearest-neighbour vertices are in yellow; triskelions centred at second nearest-neighbour vertices are in light blue and triskelions centred at third nearest-neighbour vertices are in dark blue. The auxilin fragment, outlined in red, lies between the dark blue terminal domains and the light blue ankle segments of clathrin. Hsc70, in green, binds in the funnel-like cavity bounded by these segments. The clathrin chains are in surface rendering from the molecular model; the auxilin and Hsc70 are in basket contours, based on the density. Download figure Download PowerPoint We could assign the location of Hsc70 by computing local difference maps between the reconstructions with bound auxilin (547–910) alone and with Hsc70 added (Supplementary Figure S3). These maps were computed from non-n.c.s.-averaged reconstructions, low-pass filtered to 20 Å resolution. As shown in Figure 5, difference density at each vertex abuts the C-terminus of the helical tripod, within the triangular funnel formed by three crossing ankles and three terminal domains. A short segment in the disordered region of polypeptide chain, just C-terminal to the tripod helix, has a sequence that corresponds closely to the consensus for tight binding by Hsc70 (Gragerov et al, 1994; Fotin et al, 2004a). Mutation of this segment eliminates Hsc70:ATP-dependent uncoating and substantially reduces Hsc70 binding (Rapoport et al, 2008), and we can infer that Hsc70 clamps onto the C-terminal segment at some stage during the uncoating reaction. The location of Hsc70 density suggests that it is this clamped state that we have captured—an interpretation supported by our conclusion, from the data in Figure 2, that our structure contains Hsc70 at a stage immediately following nucleotide hydrolysis. The volume and roughly three-fold symmetric shape of the Hsc70 density both suggest that we have captured a single Hsc70 at each vertex, consistent with our estimate from band strengths in Figure 1A, and that the density feature is an average from molecules in three similarly occupied orientations. Indeed, the funnel leading to the Hsc70 site is too small to accommodate more than one uncoating enzyme. In the ADP state, the two domains of Hsc70 are not fixed with respect to one another. We propose that the J-domain interacting region of Hsc70 contacts one of the three, quasi-equivalent auxilins at a vertex and that the substrate-binding domain will then be oriented to find one of the three C-terminal tails of the tripod. Conformational changes in the clathrin coat Association with uncoating factors alters the dimensions of the D6 barrel. The axial ratios shift by about 2% on binding auxilin (547–910) and by a total of about 4% on binding of both auxilin (547–910) and Hsc70 (1–554) (Figure 6A and B). Thus, the proportions of the entire barrel change when uncoating factors bind. We note that the axial ratio is insensitive to EM magnification and other scalar calibration factors. We also carried out a reconstruction of native D6 coats (in the absence of bound auxilin or Hsc70) at pH 6; the axial ratios are identical to those of native coats at pH 6.5 (Fotin et al, 2004b), and we can, therefore, rule out any purely pH-dependent (rather than uncoating-factor-dependent) mechanism. Figure 6.Conformational changes in clathrin that accompany binding of auxilin and Hsc70. (A) Axial ratios of D6 coats. The height (H) and two equatorial widths (W1 and W2), illustrated in the cartoon, are the distances between corresponding pairs of atoms at the outer margins of the molecular models. (B) Local changes in the conformation of the N-terminal parts of a triskelion in response to binding of auxilin (green) or auxilin plus Hsc70 (red). The reference triskelion is in blue. (C) Density maps and ribbon representations of a single triskelion leg from the unliganded coat (blue) and the auxilin:Hsc70-bound coat (red). Superposition determined at the hub of the triskelion, as in (B). Top: complete leg; bottom: detail of N-terminal region. The maps have been contoured generously, to show clearly the lower density of the terminal domain and linker; hence, the relatively ‘loose’ fit of the proximal and distal legs. Download figure Download PowerPoint To analyse the molecular basis for the axial-ratio change, we superposed the corresponding vertices of the three models, using as a common reference frame the three proximal–distal pairs that radiate around the vertex (Figure 6B and C). With bound uncoating factors, the crossed ankles shift radially outward to widen the opening around the foot of the helical tripod. The lever arm of the linkers, which connect into the ankle crossing from the terminal domains, amplifies the apparent shift, so that the terminal domains facing each vertex move even more noticeably away from each other. Auxilin alone appears to induce most of the change in the ankle crossing. Addition of Hsc70 widens the opening of the funnel around each tripod by enhancing the displacement of the terminal domains. How do these local shifts produce large-scale changes in axial ratio and hence generate strain in the coat? The 36 vertices of the D6 barrel fall into three symmetry-distinct classes (see Figure 1). The axial ratio depends on the relative curvature at vertices of each class. The pucker at the apex of a triskelion is invariant: the local curvature at each vertex is determined not by a change in triskelion pucker but by a change in the angle between the two proximal–distal pairs that run antiparallel to each other along an edge (Fotin et al, 2004b). The distal-leg components of each of these pairs emanate from crossed ankles at the neighbouring vertices (Figures 1 and 5). The preferred geometry of the ankle crossing, therefore, propagates into curvature preferences, because the ankle crossing at one vertex is linked by a relatively rigid structural member (the distal–proximal pair) to the three neighbouring vertices. In an isotro
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