Chaperonin complex with a newly folded protein encapsulated in the folding chamber.

A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.

Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy.

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map.

Structural diversity of ex vivo amyloid fibrils studied by cryo-electron microscopy.

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein.

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain.

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.

Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure.

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

ATP-bound states of GroEL captured by cryo-electron microscopy.

The chaperonin GroEL drives its protein-folding cycle by cooperatively binding ATP to one of its two rings, priming that ring to become folding-active upon GroES binding, while simultaneously discharging the previous folding chamber from the opposite ring. The GroEL-ATP structure, determined by cryo-EM and atomic structure fitting, shows that the intermediate domains rotate downward, switching their intersubunit salt bridge contacts from substrate binding to ATP binding domains. These observations, together with the effects of ATP binding to a GroEL-GroES-ADP complex, suggest structural models for the ATP-induced reduction in affinity for polypeptide and for cooperativity. The model for cooperativity, based on switching of intersubunit salt bridge interactions around the GroEL ring, may provide general insight into cooperativity in other ring complexes and molecular machines.

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane.

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.

Structural basis of pore formation by cholesterol-binding toxins.

In this paper we describe reconstructions by electron cryo-microscopy of two oligomeric states of the pore-forming toxin pneumolysin. The results are interpreted by the fitting of atomic models of separated domains to the 3-dimensional electron density maps, revealing two steps in the mechanism of pore formation by the family of cholesterol-binding toxins. We briefly describe the observation of the toxin pore in model membranes and contrast the apparent mechanism of pneumolysin with that of other pore-forming toxins.

Macromolecular structure determination by cryo-electron microscopy.

Recent advances in transmission electron microscopy (EM) hardware, low-temperature methods and image-processing software have made cryo-EM an important complement to X-ray crystallography and NMR for macromolecular structure determination, particularly of large assemblies. This review provides a summary of the main advances and a survey of the capabilities of this approach.

Conformational changes studied by cryo-electron microscopy.

Biological processes involving movement, such as muscle contraction or the opening of an ion channel through a membrane, are mediated through conformational changes. These changes often occur in large and flexible macromolecular complexes. Cryo-electron microscopy provides a means of capturing different conformational states of such assemblies. Even if the resulting density maps are at low resolution, they can be combined with atomic structures of subcomplexes or isolated components determined by X-ray crystallography or NMR. This review presents a brief summary of the principles and recent advances in macromolecular structure determination by cryo-electron microscopy.

Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin.

Three conformations of the thermosome, an archaeal group II chaperonin, have been determined by cryo-electron microscopy (EM). We describe an open form of the double-ring oligomer, a closed form and a bullet-shaped form with one ring open and the other closed. Domain movements have been deduced by docking atomic coordinates into the EM maps. The subunit apical domains, bearing the putative substrate binding sites, rotate about 30 degrees upwards and twist in the plane of the ring from the closed to the open conformation. The closed rings have their nucleotide binding pockets closed by the intermediate domains, but in the open rings, the pocket is accessible.

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae.

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.

Multivalent binding of nonnative substrate proteins by the chaperonin GroEL.

The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.

The black widow's versatile venom.

The structure of a tetrameric form of the spider neurotoxin, alpha-latrotoxin, has been determined by single particle cryo-electron microscopy. It reveals a pore complex with extended arms that could bind receptors involved in synaptic vesicle exocytosis.

Hsp26: a temperature-regulated chaperone.

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.

Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein.

The virus-like particles (VLPs) produced by the yeast Ty retrotransposons are structurally and functionally related to retroviral cores. Using cryo-electron microscopy (cryo-EM) and three-dimensional (3D) reconstruction, we have examined the structures of VLPs assembled from full-length and truncated forms of the capsid structural protein. The VLPs are highly polydisperse in their radius distribution. We have found that the length of the C-terminal region of the capsid structural protein dictates the T -number, and thus the size, of the assembled particles. Each construct studied appears to assemble into at least two or three size classes, with shorter C termini giving rise to smaller particles. This assembly property provides a model for understanding the variable assembly of retroviral core proteins. The particles are assembled from trimer-clustered units and there are holes in the capsid shells.

Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.