The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin
2001; Springer Nature; Volume: 20; Issue: 15 Linguagem: Inglês
10.1093/emboj/20.15.4065
ISSN1460-2075
Autores Tópico(s)Enzyme Structure and Function
ResumoArticle1 August 2001free access The ‘sequential allosteric ring’ mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin Oscar Llorca Oscar Llorca Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Jaime Martín-Benito Jaime Martín-Benito Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author Julie Grantham Julie Grantham CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Monica Ritco-Vonsovici Monica Ritco-Vonsovici CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Keith R. Willison Keith R. Willison CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author José L. Carrascosa José L. Carrascosa Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author José M. Valpuesta Corresponding Author José M. Valpuesta Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author Oscar Llorca Oscar Llorca Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Jaime Martín-Benito Jaime Martín-Benito Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author Julie Grantham Julie Grantham CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Monica Ritco-Vonsovici Monica Ritco-Vonsovici CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author Keith R. Willison Keith R. Willison CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK Search for more papers by this author José L. Carrascosa José L. Carrascosa Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author José M. Valpuesta Corresponding Author José M. Valpuesta Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Search for more papers by this author Author Information Oscar Llorca1,2, Jaime Martín-Benito1, Julie Grantham2, Monica Ritco-Vonsovici2, Keith R. Willison2, José L. Carrascosa1 and José M. Valpuesta 1 1Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain 2CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea, London, SW3 6JB UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4065-4075https://doi.org/10.1093/emboj/20.15.4065 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5′-adenylyl-imido-diphosphate), to both CCT–α-actin and CCT–β-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP–CCT-bound conformations of α-actin and β-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states. Introduction In the past decade it has become clear that, under normal growth conditions, the folding of many proteins requires the help of other proteins, named molecular chaperones, which interact with non-native structures to help them overcome kinetic barriers on the pathway to their native folds (Bukau and Horwich, 1998). Chaperonins, one of the major classes of folding-assisting proteins, have been well characterized. They are found in all living cells, being strictly required for viability. Chaperonins comprise two subclasses: Group I, found in eubacteria and endosymbiotic organelles (Bukau and Horwich, 1998; Ellis and Hartl, 1999) and Group II, found in archaea and in the cytosol of eukaryotic cells (Gutsche et al., 1999; Willison, 1999). Among the Group I chaperonins, GroEL from Escherichia coli has been most extensively studied. Its atomic structure is known (Braig et al., 1994; Xu et al., 1997) and also the low-resolution structures of many of its reaction intermediates have been obtained by cryo-electron microscopy (cryo-EM) (Roseman et al., 1996). GroEL is made up of two identical back-to-back stacked homo-heptameric rings built with a 60 kDa subunit. Apical, intermediate and equatorial domains are the main functional components of each monomer (Braig et al., 1994). GroEL requires a cofactor, GroES, to fold substrates efficiently. GroES, a homo-heptameric ring of a 10 kDa subunit (Hunt et al., 1996), interacts with the apical domains of GroEL in the presence of nucleotide. Current models for the function of GroEL propose that unfolded substrates, displaying exposed hydrophobic regions, can be bound by the hydrophobic sites of GroEL apical domains (Chen and Sigler, 1999). Upon ATP binding to the equatorial domains, GroES interacts with the substrate-bound GroEL ring and displaces it into the now hydrophilic cavity, where it has a chance to fold correctly (Bukau and Horwich, 1998; Ellis and Hartl, 1999). However, less is known about the structures and mechanisms of action of Group II chaperonins (Gutsche et al., 1999). One of its main representatives is the cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)], also termed TRiC. CCT rings consist of eight different subunits positioned in a precise arrangement (Liou and Willison, 1997; Llorca et al., 1999b; Willison, 1999; Grantham et al., 2000). The sequence differences among the CCT subunits are located mainly in their apical domains (Kim et al., 1994), suggesting some degree of specificity towards substrate binding, which is reinforced by the fact that two proteins, actin and tubulin, have been found to be the major substrates of CCT. It has been hypothesized that a link exists between the evolution of the eukaryotic cell and the folding of actins and tubulins, and that it has involved the transformation of a more simple archaeabacterial chaperonin to the highly complex CCT (Willison, 1999; Leroux and Hartl, 2000; Llorca et al., 2000; Willison and Grantham, 2001). Recently, the three dimensional (3D) reconstruction of CCT–α-actin and CCT–β-tubulin complexes has shed some light on certain structural aspects of CCT substrate recognition (Llorca et al., 1999b, 2000); CCT seems to be recognizing actin and tubulin in native-like conformations. The eukaryotic chaperonin binds each of the two topological domains of the two cytoskeletal proteins (Kabsch et al., 1990; Nogales et al., 1998) using opposite regions of the CCT ring, thus stabilizing extended and open conformations. The CCT subunits involved in this binding mode have been mapped directly by immunoelectron microscopy. The actin small domain binds to the CCTδ subunit and the large C-terminal domain to either CCTβ or CCTϵ, the pair of latter subunits binding actin with high affinity (Llorca et al., 1999b; Hynes and Willison, 2000). On the other hand, tubulin uses two possible binding arrangements involving two sets of five subunits, but with CCTβ and CCTϵ again being the subunits binding the C-terminal domain of tubulin with the highest affinity in each of the two arrangements (Llorca et al., 2000). Biochemical studies have corroborated the identity of the actin and tubulin domains involved in the binding to specific CCT subunits (Hynes and Willison, 2000; Llorca et al., 2000; Ritco-Vonsovici and Willison, 2000). In this work, 3D reconstructions of the 5′-adenylyl-imido-diphosphate (AMP-PNP)-bound forms of CCT–α-actin and CCT–β-tubulin complexes, combined with the docking of atomic structures of the two cytoskeletal proteins and immunolabelling experiments, have shed new light on the molecular mechanism of actin and tubulin folding. The results suggest that the eukaryotic chaperonin has evolved from its precursor to fold these complex eukaryotic proteins by coupling sequential changes in the apical domains of the chaperonin subunits that occur upon nucleotide binding, to concerted movements in the substrate molecules that lead to their successful folding. Results Three-dimensional structures of the AMP-PNP–CCT and AMP-PNP–CCT–α-actin complexes α-actin was chemically denatured, incubated with CCT in a diluting buffer, and then the free actin molecules were removed by size-exclusion chromatography. The non-hydrolysable ATP analog AMP-PNP was added to a concentration high enough (10 mM) to saturate all the nucleotide binding sites and mimic ATP binding (but not hydrolysis). Aliquots of this solution were vitrified and images were obtained at low temperature (−170°C). Tilted top views of CCT were processed (Figure 1A) and the particles were classified by two different independent methods (see Materials and methods) into two populations: those with an empty cavity and those having a mass inside the chaperonin cavity. The 3D reconstruction carried out with the substrate-free CCT particles incubated with AMP-PNP (AMP-PNP–CCT from now on; Figure 1B–D) shows closure of both CCT cavities compared with the open conformation of the apo-CCT structure (Llorca et al., 1999a, 2000). The conformation obtained here is very similar to that of the thermosome X-ray structure (Ditzel et al., 1998), and the docking of the two structures reveals a good correlation (Figure 1E and F), which supports the notion that the conformation obtained for the thermosome crystal structure might be the one generated upon ATP binding (Ditzel et al., 1998; Gutsche et al., 2000). The conformation of both CCT rings is similar to one of the rings of the 3D reconstruction of the ATP-bound form of CCT [see the top ring in Figure 3A–C in Llorca et al. (1999a)], albeit at higher resolution. In the present CCT structure, the mass corresponding to the entire helical protrusions is now visualized (revealed in the docking top view; Figure 1E). In the former volumes (Llorca et al. 1999a), only the base of the apical domains was visualized, resulting in an apparent wider hole, whereas the structure revealed here shows the real magnitude of the closure [compare Figure 5A of Llorca et al. (1999a) with Figure 1E of this work]. The differences between the two 3D reconstructions have to do not only with the improvement in the image processing, but also to the fact that the latter reconstruction has been carried out using a more homogeneous population (generated with the non-hydrolysable analog AMP-PNP) than the previous one (generated using ATP and possibly having a mixture of conformations). Figure 1.Three-dimensional reconstruction of CCT in the presence of AMP-PNP. (A) A gallery of ice-embedded CCT particles in the presence of AMP-PNP. (B–D) Different views of the 3D structure of the AMP-PNP–CCT complex, generated after the processing of 1057 particles. (E and F) Top and side views, respectively, of the docking between the thermosome X-ray structure (in purple and white; Ditzel et al., 1998) and the 3D reconstruction of AMP-PNP–CCT (yellow grid). Download figure Download PowerPoint Figure 2.Three-dimensional reconstruction of CCT–α-actin complex in the presence of AMP-PNP. (A–C) Different views of the 3D structure of the AMP-PNP–CCT–α-actin complex generated after the processing of 1640 particles. (C) A cut along the longitudinal axis whereby the substrate in the interior of the cavity is revealed. (D and E) The transparent AMP-PNP–CCT–α-actin volume showing the actin molecule, calculated by subtracting the volumes of the actin-containing ring and the substrate-free ring, shaded in red. (F) Top and side views of the extracted actin volume. Download figure Download PowerPoint Figure 3.Three-dimensional reconstruction of the CCT–β-tubulin complex in the presence of AMP-PNP. (A and B) Different views of the AMP-PNP–CCT–β-tubulin complex, generated after the processing of 3501 particles. In (B), a cut along the longitudinal axis shows the tubulin molecule in the interior of the cavity. (C) Side view of the docking between the thermosome X-ray structure (in purple and white; Ditzel et al., 1998) and the 3D reconstruction of AMP-PNP–CCT–tubulin (yellow grid). (D and E) The transparent AMP-PNP–CCT–β-tubulin volume with the tubulin molecule, calculated by subtracting the volumes of the tubulin-containing ring and the substrate-free ring, shaded in red. (F) Two views of the extracted tubulin volume. Download figure Download PowerPoint The 3D structure of the CCT–α-actin complex in the presence of AMP-PNP (AMP-PNP–CCT–α-actin complex from now on) is very similar to the substrate-free structure described above, but shows an extra density close to the apical regions of one of the rings (Figure 2C). As occurs during the 3D reconstruction of the nucleotide-free CCT–α-actin complex (Llorca et al., 1999b), no particles of CCT have been found containing substrate molecules in both rings. The reconstructed volume of the actin molecule can be determined from the bulk of the reconstruction after calculation of the difference map between the actin-bound and actin-free ring of the 3D reconstruction (red shading in Figure 2D and E). A similar volume is obtained after calculating the difference map between the actin-bound ring of the AMP-PNP–CCT–α-actin complex and either of the two rings of the CCT–AMP-PNP complex (not shown). The volume of the actin molecule extracted from the 3D reconstruction (top and side view in Figure 2F and G, respectively) shows a heart-like shape (Figure 2F) similar to the atomic structure of actin (Kabsch et al., 1990) when filtered at low resolution. Parenthetically, the actin molecule seems attached to the apical domains and not liberated into the cavity. Three-dimensional structure of the AMP-PNP–CCT–β-tubulin complex Recombinant human β-tubulin was chemically denatured, incubated with CCT in a diluting buffer, and unbound tubulin was subsequently removed by size-exclusion chromatography. Upon incubation with AMP-PNP, cryo-EM of the CCT particles was performed. Tilted top views of CCT were processed and classified according to the presence or absence of mass inside the chaperonin cavity. The 3D reconstruction of the β-tubulin-bound CCT particles shows a very symmetrical, closed structure (Figure 3A and B) with the tubulin molecule placed off-centre and hanging from the apical domains of one of the rings (Figure 3B and red shading in D and E). The degree of closure of both rings is greater than that observed for the 3D reconstructions of both the apo-CCT and the CCT–α-actin complex in the presence of AMP-PNP (Figures 1 and 2). It has been shown previously that the nucleotide-free conformation of the CCT–β-tubulin complex reveals a more closed disposition of the rings than in the case of the apo-CCT or the CCT–α-actin complex (Llorca et al., 2000), and this behaviour seems to be continued in the nucleotide-bound state. This is confirmed when a docking between the atomic structure of the thermosome and the 3D structure of the AMP-PNP–CCT–β-tubulin complex is performed (Figure 3C), which again reveals a good fitting between the two structures, except that, in contrast to the docking carried out with the AMP-PNP–CCT structure (Figure 1E and F) or the AMP-PNP–CCT–α-actin complex (not shown), the apical domains of the AMP-PNP–CCT–β-tubulin complex are pulled inwards and downwards (∼8 Å; see top and bottom of Figure 3C). The most likely explanation for this difference in the CCT conformations generated by the two cytoskeletal proteins is that whereas actin interacts with CCT subunits through two delimited regions (Llorca et al., 1999b), tubulin binds to five different CCT subunits (Llorca et al., 2000) via apparently numerous binding sites (Ritco-Vonsovici and Willison, 2000), thus generating a more compact structure. Another notable structural feature observed in the 3D reconstruction of the AMP-PNP–CCT-β-tubulin complex presented here and in the nucleotide-free CCT–β-tubulin complex shown in Llorca et al. (2000) is that the downward and inward movements of the apical domains take place simultaneously in both rings, despite the fact that only one of the rings contains tubulin. This symmetry is also found in the atomic structure of the thermosome (Ditzel et al., 1998) and in the structures of the thermosome and TF55 determined by cryo-EM by Schoehn et al. (2000a,b), and may reflect the very strong negative co-operativity between CCT rings upon ATP binding, since the Hill coefficient is practically equivalent to the number of subunits in the ring (7.2; Kafri et al., 2001). A possible explanation for this behaviour may be due to the differences in the inter-ring subunit arrangement between Group I chaperonins, in which every subunit interacts with two opposite subunits (Braig et al., 1994), and Group II chaperonins, in which each subunit opposes only one subunit (Ditzel et al., 1998). As mentioned above, the tubulin mass is found hanging from the apical domains of one side of the CCT ring. The tubulin mass can be extracted from the bulk of the CCT volume by calculating the difference map between the tubulin-loaded ring and the substrate-free ring of the CCT–β-tubulin complex. The extracted tubulin (Figure 3F and red shading in D and E) has a compact shape and occupies approximately one-third of the cavity volume (Figure 3B). The fact that the reconstructed volume of tubulin is located off-centre, interacting with one side of the CCT cavity and only partially occupying it, precludes the possibility of the reconstructed substrate coming from the averaging of tubulin molecules randomly placed within the cavity. As in the case of actin, the structural rearrangements occurring in the CCT–β-tubulin complex upon nucleotide binding close the chaperonin cavity, but tubulin continues to be bound to the apical domains. Biochemical correlates of the closure of the CCT cavity upon nucleotide binding We wanted to confirm the closure of the CCT cavity upon nucleotide binding by using a biochemical technique, and for this we used differential immunoprecipitation of CCT by binding monoclonal antibodies reacting to different regions of the chaperonin (Figure 4). Immunoprecipitation of [35S]CCT in the absence or presence of AMP-PNP with the monoclonal antibody 23C, which binds to the very C-terminus of CCTα, located inside the chaperonin cavity (Grantham et al., 2000), shows that CCT–AMP-PNP virtually does not immunoprecipitate with 23C. This is consistent with the model that the helical protrusions obscure the CCT cavity upon nucleotide binding and block the access of the antibody to the chaperonin cavity. On the other hand, the variation in immunoprecipitation yield of [35S]CCT in the absence or presence of AMP-PNP with the monoclonal antibody ϵAD1 is much less perturbed. ϵAD1 recognizes an epitope on the outer surface of the helical protrusion of the CCTϵ apical domain, when compared with the atomic structure of the thermosome (both α- and β-subunits) (Ditzel et al., 1998), and would be expected to be able to bind CCT in the presence and absence of nucleotide. Figure 4.Immunoprecipitation of CCT in the presence of AMP-PNP. Sucrose gradient (20S) samples containing [35S]CCT were incubated in the presence and absence of 10 mM AMP-PNP, and immunoprecipitated under non-denaturing conditions with either a C-terminus anti-CCTα or an anti-CCTϵ apical domain monoclonal antibody. Recovered proteins were analysed by SDS–PAGE followed by autoradiography. The starting material (S) and background (minus antibody) controls are indicated. Download figure Download PowerPoint Figure 5.Docking of the atomic coordinates of actin and tubulin with their corresponding EM volumes extracted from the AMP-PNP–CCT–α-actin and AMP-PNP–CCT–β-tubulin complexes. (A and B) Two views of the docking with actin. (C and D) Two views of the docking with tubulin. In both cases, the envelope of the EM volume has been drawn as a yellow grid. Different domains of the actin and tubulin structures have been coloured as in Llorca et al. (2000). For α-actin, N-terminal domain residues D1–R177 are coloured red and the C-terminal residues Q263–R372 and L178–F262 yellow and white, respectively. For β-tubulin, the N-terminal residues M1–I204 are coloured red, and the C-terminal residues D205–L265, H266–I384 and Q385–D437 are coloured green, white and yellow, respectively. Nucleotides (ATP for actin and GTP for tubulin) are coloured blue. Download figure Download PowerPoint Docking of the actin and tubulin X-ray coordinates within their reconstructed volumes The volumes of α-actin and β-tubulin are not averaged out during the 3D reconstruction procedure, clearly indicating that the two cytoskeletal proteins maintain their interaction with CCT after nucleotide binding, each probably in a native or quasi-native conformation. To test this hypothesis, the atomic coordinates of the native structures of actin (Kabsch et al., 1990) and tubulin (Nogales et al., 1998) were fitted, using the SITUS quantitative algorithm (Wriggers et al., 1999), into the volumes of the EM counterparts extracted from their respective complexes. The reconstructed volume of the α-actin molecule bound to CCT fits well with its native X-ray structure (Figure 5A and B), and there is a good agreement between the different actin subdomains and the lobular structure of the reconstructed volume (see Figure 2F and G). The two best solutions in terms of root-mean square (r.m.s.) generated by the docking algorithm, and both of them being significantly better than any of the others obtained, place the atomic structure of actin horizontally into the flat shape of the reconstructed volume of actin and with one of the tips of the actin molecule (Kabsch et al., 1990) interacting with an apical domain of CCT. This uncertainty probably has to do with the low resolution of the reconstructed actin. One of the solutions places the actin molecule with the tip of the large domain interacting with the CCT apical domains. This actin region is the same one that has been shown to bind to the eukaryotic chaperonin in its nucleotide-free form [white domain in Figure 5A and B; see the same domain in Llorca et al. (2000)]. The second solution, however, places this actin region pointing towards the CCT cavity and not interacting with the apical domains. If this second solution were correct, this would imply that after nucleotide binding to CCT, the actin molecule would lose all its contacts with the apical domains of CCT and rebind to the chaperonin using completely different regions. Since the first solution maintains the tip of the large domain, the region that has been shown to bind CCT with the highest affinity (Rommelaere et al., 1999; Hynes and Willison, 2000; Llorca et al., 2000), interacting with CCT, it accounts, in a simpler and more reasonable way, for the transition between the nucleotide-free and the nucleotide-bound conformation by preserving the interactions between the chaperonin and the respective substrate. The atomic structure of native tubulin also fits quite well into the volume of the reconstructed tubulin complexed to nucleotide-bound CCT (Figure 5C and D). The two best solutions provided by the docking algorithm, both significantly better than any others, place the atomic structure of tubulin in a vertical position, hanging from the underside of the apical domains. In the first of the two solutions, the region that the docking algorithm places as interacting with CCT is the one possessing the highest affinity for apo-CCT [white domain in Figure 5C and D; see the same domain in Llorca et al. (2000) and Ritco-Vonsovici and Willison (2000)]. The other solution places the tubulin molecule upside down with the highest affinity binding region pointing to the centre of the cavity. This uncertainty may again have to do with the low resolution of the reconstructed tubulin. As discussed previously in the case of AMP-PNP–CCT–α-actin, the first solution seems to maintain the tubulin molecule bound to the chaperonin using the same CCT-binding regions before and after the nucleotide binding, and therefore has been chosen as the most likely possibility. This assumption correlates well with the immunolabelling experiments described below. Immunoelectron microscopy of the CCT–β-tubulin complex in the presence of AMP-PNP After careful inspection of the tubulin location in the AMP-PNP–CCT–β-tubulin complex (Figure 3), it becomes clear that the substrate is placed asymmetrically in the chaperonin cavity (see in Figure 6A the projection of the AMP-PNP–CCT–β-tubulin complex along the longitudinal axis). Based on the previous findings, which locate the tubulin molecule bound to specific CCT subunits before nucleotide binding to the chaperonin (Llorca et al., 2000), we reasoned that tubulin could also be placed in a defined position within the chaperonin cavity after nucleotide binding. To prove this hypothesis, we labelled the AMP-PNP–CCT–β-tubulin complex with a monoclonal antibody specifically recognizing the CCTδ subunit. The average image obtained after the processing of two-dimensional (2D), negatively stained, top views of the complexes (Figure 6B) clearly shows that the tubulin density is placed almost opposite to the labelled CCTδ subunit and near the CCTβ/ϵ region. It has previously been shown (Llorca et al., 2000) that tubulin binds to CCT in two different geometrical arrangements involving the interaction of the C-terminal region of tubulin with the CCTϵ/ζ/β or CCTβ/γ/σ subunits, and the N-terminal domain with the diametrically opposite subunits (CCTσ/δ or CCTη/α subunits, respectively). The average image of the immunocomplex presented here clearly suggests that, after nucleotide binding, the tubulin molecule is displaced away from the CCTδ subunit and moves towards the CCTβ/ϵ subunits, the subunits involved in binding tubulin with the highest affinity in the previous step of the folding cycle (Llorca et al., 2000; Ritco-Vonsovici and Willison, 2000). Figure 6.Immunomicroscopy of the AMP-PNP–CCT–β-tubulin complex incubated with anti-CCTδ. (A) Projection of the 3D reconstruction of the AMP-PNP–CCT–β-tubulin complex along the z-axis. (B) Two-dimensional average image of the top views obtained from negatively stained immunocomplexes of AMP-PNP–CCT–β-tubulin–8g (anti-CCTδ) (405 particles). Note that the different shape of both images is due to the fact that (A) is the projection of the whole CCT structure, whereas (B) is the projection of the apical domains of the grid-interacting ring. Download figure Download PowerPoint A similar approach could not be carried out with actin because the reconstructed molecule is placed horizontally, filling the upper part of the cavity completely, making it impossible to distinguish different locations within the cavity by 2D immunomicroscopy. Discussion Conformational changes of CCT upon ATP binding and hydrolysis As with Group I chaperonins, ATP-driven conformational changes are essential to the mechanism of the eukaryotic CCT (Gutsche et al., 1999; Willison, 1999). Despite their great mechanistic importance, there are still discrepancies and uncertainties about the effects of different nucleotides on Group II chaperonin structure. For the archaeal chaperonins, open, closed and bullet-shaped conformations have been found by cryo-EM and 3D reconstruction (Schoehn et al., 2000a,b), but their occurrence is, according to these authors, independent of the presence or absence of nucleotides. However, small-angle neutron scattering experiments carried out on the thermosome suggest that ATP binding induces an even wider opening of the cavity, which is closed only after nucleotide hydrolysis (ADP-Pi conformation; Gutsche et al., 2000). The X-ray structure of the nucleotide-fre
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