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.
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.
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.
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 structure of bovine F 1 -ATPase inhibited with ADP and beryllium fluoride at 2.0 Å resolution contains two ADP.BeF 3 À complexes mimicking ATP, bound in the catalytic sites of the b TP and b DP subunits. Except for a 1 Å shift in the guanidinium of aArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the c-phosphate of ATP during hydrolysis is 2.6 Å from the beryllium in the b DP subunit and 3.8 Å away in the b TP subunit, strongly indicating that the b DP subunit is the catalytically active conformation. In the structure of F 1 -ATPase with five bound ADP molecules (three in a-subunits, one each in the b TP and b DP subunits), which has also been determined, the conformation of aArg373 suggests that it senses the presence (or absence) of the cphosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F 1 -ATPase.
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.
Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATPdependent 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" =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. (Hsp60) (13), and the second family with members in thermophilic archaebacteria (TF55) and the eukaryotic cytosol (TCP1 complex) (14, 15).Studies carried out both in intact cells and with the purified chaperonin complexes in vitro suggest that binding of unfolded proteins by these components prevents aggregation and maintains the bound proteins in productive intermediate conformations that are at least in some cases collapsed "molten globule"-like forms (16-26). In the presence of cochaperonin and ATP hydrolysis, bound polypeptides are released from chaperonin complexes associated with folding to native conformation. At least the initial stages of folding appear to occur while the polypeptide chain remains associated with the complex (21).Little is known about the nature of interaction between unfolded polypeptides and chaperonin complexes. A fundamental question concerns where polypeptides localize in relation to the double ring structures. Here we have addressed this question by direct inspection in the scanning transmission electron microscope (STEM) of a polypeptide tagged with a gold cluster bound to the Escherichia coli chaperonin groEL. MATERIALS AND METHODSCoupling Reactions and Methotrexate-Agarose Affinity Chromatography. Gold clusters (200 nmol; diameter, 1.4 nm) (27) were incubated with 20 mmol of iodoacetic acid N-hydroxysuccinimide ester for 45 m...
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.
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