Chaperonins.

Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric GroEL-GroES complex and of their isolated peptide-binding domains.

Bovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.

BACKGROUND: F1-ATPase is the globular domain of F1F0-ATP synthase that catalyses the hydrolysis of ATP to ADP and phosphate. The crystal structure of bovine F1-ATPase has been determined previously to 2.8 A resolution. The enzyme comprises five different subunits in the stoichiometry alpha 3 beta 3 gamma delta epsilon; the three catalytic beta subunits alternate with the three alpha subunits around the centrally located single gamma subunit. To understand more about the catalytic mechanisms, F1-ATPase was inhibited by reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and the structure of the inhibited complex (F1-NBD) determined by X-ray crystallography. RESULTS: In the structure the three beta subunits adopt a different conformation with different nucleotide occupancy. NBD-Cl reacts with the phenolic oxygen of Tyr311 of the beta E subunit, which contains no bound nucleotide. The two other catalytic subunits beta TP and beta DP contain bound adenylyl-imidodiphosphate (AMP-PNP) and ADP, respectively. The binding site of the NBD moiety does not overlap with the regions of beta E that form the nucleotide-binding pocket in subunits beta TP and beta DP nor does it occlude the nucleotide-binding site. Catalysis appears to be inhibited because neither beta TP nor beta DP can accommodate a Tyr311 residue bearing an NBD group. CONCLUSIONS: The results presented here are consistent with a rotary catalytic mechanism of ATP synthesis and hydrolysis, which requires the sequential and concerted participation of all three catalytic sites. NBD-Cl inhibits the enzyme by preventing the modified subunit from adopting a conformation that is essential for catalysis to proceed.

Structure of bovine mitochondrial F(1)-ATPase inhibited by Mg(2+) ADP and aluminium fluoride.

BACKGROUND: The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. RESULTS: The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein. CONCLUSIONS: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.

Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution.

Improved refinement of the crystal structure of GroEL from Escherichia coli has resulted in a complete atomic model for the first 524 residues. A new torsion-angle dynamics method and non-crystallographic symmetry restraints were used in the refinement. The model indicates that conformational variability exists due to rigid-body movements between the apical and intermediate domains of GroEL, resulting in deviations from strict seven-fold symmetry. The regions of the protein involved in polypeptide and GroES binding show unusually high B factors; these values may indicate mobility or discrete disorder. The variability of these regions may play a role in the ability of GroEL to bind a wide variety of substrates.

The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL.

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements. Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES. Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 A resolution for GroEL and GroEL-GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP. Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein-binding sites. We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

A polypeptide bound by the chaperonin groEL is localized within a central cavity.

Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state. Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter. We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters. In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings. In some cases, two gold densities were observed in the same groEL complex. We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings. In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.

The crystal structure of the bacterial chaperonin GroEL at 2.8 A.

The crystal structure of Escherichia coli GroEL shows a porous cylinder of 14 subunits made of two nearly 7-fold rotationally symmetrical rings stacked back-to-back with dyad symmetry. The subunits consist of three domains: a large equatorial domain that forms the foundation of the assembly at its waist and holds the rings together; a large loosely structured apical domain that forms the ends of the cylinder; and a small slender intermediate domain that connects the two, creating side windows. The three-dimensional structure places most of the mutationally defined functional sites on the channel walls and its outward invaginations, and at the ends of the cylinder.

Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES.

The chaperonin GroEL is a large, double-ring structure that, together with ATP and the cochaperonin GroES, assists protein folding in vivo. GroES forms an asymmetric complex with GroEL in which a single GroES ring binds one end of the GroEL cylinder. Cross-linking studies reveal that polypeptide binding occurs exclusively to the GroEL ring not occupied by GroES (trans). During the folding reaction, however, released GroES can rebind to the GroEL ring containing polypeptide (cis). The polypeptide is held tightly in a proteolytically protected environment in cis complexes, in the presence of ADP. Single turnover experiments with ornithine transcarbamylase reveal that polypeptide is productively released from the cis but not the trans complex. These observations suggest a two-step mechanism for GroEL-mediated folding. First, GroES displaces the polypeptide from its initial binding sites, sequestering it in the GroEL central cavity. Second, ATP hydrolysis induces release of GroES and productive release of polypeptide.

The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis.

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.