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Chaperonin-Mediated Protein Folding
Summary: Arthur Horwich is interested in understanding how chaperonin ring machines assist protein folding in the cell, and in how protein misfolding causes neurodegenerative disease.
The final step of information transfer from DNA to effector protein involves the proper folding of a newly translated polypeptide into its biologically active, native form. Although all of the information necessary to direct this process is contained in the sequence of amino acid–building blocks that are strung together to make a polypeptide chain, there is a class of specialized proteins known as molecular chaperones that provide assistance to the folding process in the intact cell, in particular, preventing polypeptide chains from misfolding and aggregating with each other. The common feature recognized by molecular chaperones is exposed hydrophobic surface, which is ultimately buried in the interior of the folded, native protein. These exposed surfaces cause nonnative proteins to stick to each other, causing multimolecular aggregation, a state that carries no biological activity and that also can be damaging to the cell, as in a number of neurodegenerative diseases. Chaperones prevent such aggregation from occurring by proffering their own hydrophobic binding sites, capturing nonnative proteins. Following release, a chaperoned protein has a fresh chance to fold properly. We have been focusing on one type of chaperone, called a chaperonin, a double-ring "machine" that has the remarkable ability to promote folding of an initially captured protein to its native state inside an encapsulated chamber.
We originally discovered chaperonin-mediated folding in mitochondria when we uncovered an action of the mitochondrial chaperonin, called heat-shock protein 60 (Hsp60), that supports the folding of newly imported proteins. We identified structurally related relatives of Hsp60 in the bacterial cytoplasm (GroEL) and chloroplast and observed more distant relatives in archaebacteria and the cytosol of higher organisms. We have directed our work toward understanding how these machines work, focusing on the bacterial machine, GroEL. Analogous to Hsp60, GroEL assists the folding of newly translated proteins in the bacterial cytoplasm. Binding and coprecipitation experiments have identified a considerable cohort of protein species that associate with the chaperonin. In addition, we have recently examined the effects of a severe temperature-sensitive lethal mutant affecting GroEL and observed extensive aggregation at the nonpermissive temperature, affecting a broad collective of newly translated cytosolic proteins of all sizes. This suggests that either aggregation is occurring as an "avalanche" effect, with a few misfolding proteins entraining the remaining newly translated ones, or that most newly made cytosolic proteins interact with GroEL at least transiently to achieve proper folding to their active native state. To resolve these possibilities, we are testing the direct physical interaction of newly translated proteins with GroEL in the living cell.
To dissect the mechanism by which GroEL assists folding, we have used a variety of techniques, including biochemical studies, x-ray crystallography (carried out with the late Paul Sigler), cryoEM studies (with Helen Saibil [Birkbeck College, London]), and NMR studies (with Kurt Wüthrich [Swiss Federal Institute of Technology, ETH, Zürich]). This has given us a working model of GroEL action, involving assistance by a small cochaperonin "lid," GroES, and consumption of ATP (see the figure for a schematic illustration of this model).
Two Steps of Chaperonin Assistance: Polypeptide Binding by an Open Ring, and Folding in an Encapsulated Chamber
Binding. GroEL is a back-to-back arrangement of two seven-membered rings, with a large central cavity at either end. This is the site of binding nonnative protein. The inside of this cavity is lined, at its apical end, with hydrophobic residues that are essential for binding nonnative proteins. Such binding in many cases involves interaction between the nonnative protein and several surrounding apical domains. It is such multivalent capture by this surface of GroEL that prevents a nonnative protein from misfolding and aggregation. It may also exert an unfolding action that takes misfolded states apart, giving them a fresh chance to subsequently fold correctly. Current deuterium-exchange studies are addressing whether this occurs on the normal timescale of the reaction (see the figure), where a ring is available for binding for about 1 second before it becomes bound by GroES. Supporting the possibility of an unfolding action are physical measurements taken on proteins stably bound by GroEL over many minutes, where no organized structure is observed.
Folding. Farther down the central cavity, at the equatorial level of the GroEL cylinder, each subunit contains an ATP-binding pocket. Binding and hydrolysis of ATP in these sites drive the cycle of binding and release of polypeptide and GroES (see the figure). Our earlier studies showed that the binding of ATP and GroES to the same ring as polypeptide (i.e., in cis) releases the polypeptide into a now GroES-encapsulated cavity where folding commences. The cavity environment is privileged. For one thing, the polypeptide folds in solitary confinement—there is no other protein with which it can aggregate. Furthermore, the walls of the chamber are switched to hydrophilic character by rigid body movements of the apical domains that occur upon ATP/GroES binding. The presence of such a hydrophilic cavity wall favors that the folding protein bury its exposed hydrophobic surfaces and expose its hydrophilic ones, properties of the native state. Whether the cavity walls participate in any more active way in assisting productive folding is currently under study. Notably, we have not observed that "stuffing" the cavity produces any increase in the rate of folding of GroEL-GroES–dependent substrates. Also, a recent experiment monitoring the trajectory of folding of the enzyme dihydrofolate reductase in the cavity shows no difference from the trajectory free in solution. The recovery of native enzyme is greater, however, in the cavity, whereas in solution there is substantial loss to aggregation.
The Neurodegenerative Disease ALS
We have also begun to investigate a neurodegenerative condition, Lou Gehrig's disease, also known as ALS (amyotrophic lateral sclerosis), in which progressive loss of motor function occurs in association with cytosolic aggregates in motor neurons. We have focused on one form of this condition associated with inherited mutation affecting the cytosolic enzyme superoxide dismutase (SOD1). We are modeling the condition by producing Caenorhabditis elegans worms or mice that are transgenic for a human ALS-causing mutant SOD. This mutant form of SOD produces a misfolded SOD protein, whose effects we are studying using genetic tools in the worm system and a variety of imaging and proteomic tools in the mouse system.
Grants from the National Institutes of Health provided support for a portion of these projects.
Last updated: February 14, 2008
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