The chaperonin CCT controls T cell receptor-driven 3D configuration of centrioles.

T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, Δ1 and Δ2) of αß-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.

Single centrosome manipulation reveals its electric charge and associated dynamic structure.

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.

Purification and functional characterization of p16, the ATPase of the bacteriophage Phi29 packaging machinery.

Bacteriophage Phi29 codes for a protein (p16) that is required for viral DNA packaging both in vivo and in vitro. Co-expression of p16 with the chaperonins GroEL and GroES has allowed its purification in a soluble form. Purified p16 shows a weak ATPase activity that is stimulated by either DNA or RNA, irrespective of the presence of any other viral component. The stimulation of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Phi29 enzyme is similar to other viral terminases. Protein p16 competes with DNA and RNA in the interaction with the viral prohead, which occurs through the N-terminal region of the connector protein (p10). In fact, p16 interacts in a nucleotide-dependent fashion with the viral Phi29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA. Our results are consistent with a model for DNA translocation in which p16, bound and organized around the connector, acts as a power stroke to pump the DNA into the prohead, using the hydrolysis of ATP as an energy source.

HYDROMIC: prediction of hydrodynamic properties of rigid macromolecular structures obtained from electron microscopy images.

We have developed a procedure for the prediction of hydrodynamic coefficients and other solution properties of macromolecules and macromolecular complexes whose volumes have been generated from electron microscopy images. Starting from the structural files generated in the three-dimensional reconstructions of such molecules, it is possible to construct a hydrodynamic model for which the solution properties can be calculated. We have written a computer program, HYDROMIC, that implements all the stages of the calculation. The use of this procedure is illustrated with a calculation of the solution properties of the volume of the cytosolic chaperonin CCT, obtained from cryoelectron microscopy images.

Point mutations in a hinge linking the small and large domains of beta-actin result in trapped folding intermediates bound to cytosolic chaperonin CCT.

The 30-A cryo-EM-derived structure of apo-CCT-alpha-actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCTbeta-CCTdelta or CCTepsilon-CCTdelta, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137-S145 and P333-S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of beta-actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three-dimensional reconstruction of the substrate-bound ring of the apo-CCT-beta-actin complex shows that beta-actin G150P is not able to bind across the chaperonin cavity to interact with the CCTdelta subunit. beta-actin G150P seems tightly packed and apparently bound only to the CCTbeta and CCTepsilon subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.

Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin.

Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins. The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins. GroEL uses a nonspecific mechanism by which any conformation of practically any unfolded polypeptide interacts with it through exposed, hydrophobic residues. ATP binding liberates the substrate in the GroEL cavity where it is given a chance to fold. A second mechanism has been described for the eukaryotic chaperonin CCT, which interacts mainly with the cytoskeletal proteins actin and tubulin. Cryoelectron microscopy and biochemical studies have revealed that both of these proteins interact with CCT in quasi-native, defined conformations. Here we have performed a detailed study of the docking of the actin and tubulin molecules extracted from their corresponding CCT:substrate complexes obtained from cryoelectron microscopy and image processing to localize certain regions in actin and tubulin that are involved in the interaction with CCT. These regions of actin and tubulin, which are not present in their prokaryotic counterparts FtsA and FtsZ, are involved in the polymerization of the two cytoskeletal proteins. These findings suggest coevolution of CCT with actin and tubulin in order to counteract the folding problems associated with the generation in these two cytoskeletal protein families of new domains involved in their polymerization.

The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin.

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-alpha-actin and CCT- beta-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 alpha-actin and beta-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.

Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle.

A three-dimensional structural model of an influenza virus ribonucleoprotein particle reconstituted in vivo from recombinant proteins and a model genomic vRNA has been generated by electron microscopy. It shows a circular shape and contains nine nucleoprotein monomers, two of which are connected with the polymerase complex. The nucleoprotein monomers show a curvature that may be responsible for the formation of helical structures in the full-size viral ribonucleoproteins. The monomers show distinct contact boundaries at the two sides of the particle, suggesting that the genomic RNA may be located in association with the nucleoprotein at the base of the ribonucleoprotein complex. Sections of the three-dimensional model show a trilobular morphology in the polymerase complex that is consistent with the presence of its three subunits.

Structural comparison of prokaryotic and eukaryotic chaperonins.

Chaperonins are key components of the cell machinery and are involved in the productive folding of proteins. Most chaperonins share a common general morphology based in a cylinder composed of two rings of 7-9 subunits, with a conspicuous cavity inside the particle. Chaperonins have been classified into two groups according to their sequence homologies: type I, whose better known member is GroEL, and type II comprising the eukaryotic cytosolic CCT and the archaebacterial thermosome, among others. Although the basic structure of both chaperonin types is rather similar, there are a number of basic differences among them. Whereas GroEL is rather non-specific regarding its substrate, CCT is more specialized, and plays a fundamental role in the folding of cytoskeletal proteins. Another important difference is that GroEL is an homopolymer, while CCT is an heteromeric complex built up of eight different polypeptides. Furthermore, GroEL requires a cofactor (GroES) that is not present in the type II chaperonins. Recent studies of the structure of CCT have allowed a deeper insight into its function. Electron microscopic analyses have revealed a different behavior of this chaperonin after binding to nucleotides, respect to GroEL. The atomic structure of the thermosome fits into the electron microscopy reconstructed volume of the CCT. This fitting gives clues to compare the structural transitions of GroEL and CCT during the folding cycle. The different changes undergone by the two chaperonins suggest the existence of differences in the way they bind substrates and enlarge the internal cavity, as well as a different type of signaling between the two rings of the types I and II chaperonins.

Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study.

We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.

Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations.

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the alpha and beta isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.

Structural analysis of the bacteriophage T3 head-to-tail connector.

The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli. Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers. The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system. Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals. The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P42(1)2 space group, with cell unit dimensions of 165 x 165 A. The three-dimensional reconstruction generated with images of crystals ranging from 0 degrees to 60 degrees tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage. A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead. The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function.

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study.

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.

Topology of the components of the DNA packaging machinery in the phage phi29 prohead.

Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role. We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector. The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead. Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity. The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector.

Partial occlusion of both cavities of the eukaryotic chaperonin with antibody has no effect upon the rates of beta-actin or alpha-tubulin folding.

The eukaryotic chaperonin containing T-complex polypeptide 1 (CCT) is required in vivo for the production of native actin and tubulin. It is a 900-kDa oligomer formed from two back-to-back rings, each containing eight different subunits surrounding a central cavity in which interactions with substrates are thought to occur. Here, we show that a monoclonal antibody recognizing the C terminus of the CCTalpha subunit can bind inside, and partially occlude, both cavities of apo-CCT. Rabbit reticulocyte lysate was programmed to synthesize beta-actin and alpha-tubulin in the presence and absence of anti-CCTalpha antibody. The binding of the antibody inside the cavity and its occupancy of a large part of it does not prevent the folding of beta-actin and alpha-tubulin by CCT, despite the fact that all the CCT in the in vitro translation reactions was continuously bound by two antibody molecules. Furthermore, no differences in the protease susceptibility of actin bound to CCT in the presence and absence of the monoclonal antibody were detected, indicating that the antibody molecules do not perturb the conformation of actin folding intermediates substantially. These data indicate that complete sequestration of substrate by CCT may not be required for productive folding, suggesting that there are differences in its folding mechanism compared with the Group I chaperonins.

Ultrastructural and functional analyses of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification.

Influenza virus ribonucleoproteins (RNPs) were reconstituted in vivo from cloned cDNAs expressing the three polymerase subunits, the nucleoprotein (NP), and short template RNAs. The structure of purified RNPs was studied by electron microscopy and image processing. Circular and elliptic structures were obtained in which the NP and the polymerase complex could be defined. Comparison of the structure of RNPs of various lengths indicated that each NP monomer interacts with approximately 24 nucleotides. The analysis of the amplification of RNPs with different lengths showed that those with the highest replication efficiency contained an even number of NP monomers, suggesting that the NP is incorporated as dimers into newly synthesized RNPs.

Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation.

TrwB is the conjugative coupling protein of plasmid R388. TrwBDeltaN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBDeltaN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K(s) = 8.7 microM) but did not show measurable ATPase or GTPase activity. A single ATP binding site was found per TrwB monomer. An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBDeltaN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBDeltaN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA around nic. The mutant protein TrwB(K136T)DeltaN70 was indistinguishable from TrwBDeltaN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits.

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.

The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 A resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity.