The Structure of ClpB. A Molecular Chaperone that Rescues Proteins from an Aggregated State
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA. Cell
(Impact Factor: 32.24).
Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.
Available from: Clarisse Brígido
- "The bacterial ClpB monomer contains an N-terminal domain and two AAA+ nucleotide-binding domains (NBD) separated by a coiled-coil middle-domain (M-domain) (Barnett et al. 2000; DeSantis and Shorter 2012; Doyle and Wickner 2009). Several studies indicate that the M-domain is essential for the chaperone activity (Barnett et al. 2005; Kedzierska et al. 2003; Lee et al. 2005; Mogk et al. 2003; Schirmer et al. 2004). Protein disaggregation by ClpB in vitro requires the collaboration of a second ATP-dependent molecular chaperone, DnaK, to promote the solubilization and reactivation of proteins that misfold and aggregate following heat shock (Goloubinoff et al. 1999; Motohashi et al. 1999; Zolkiewski 1999). "
- "ClpB monomers are arranged into homohexamers that constitute the protein functional unit. Each monomer is composed by an N-terminal domain that improves the reactivation efficiency of stable protein aggregates, two nucleotide-binding domains (NBD1 and NBD2) that bind and hydrolyze ATP, and a middle M domain that is inserted into the NBD1 and is strictly required for the disaggregase activity of the chaperone (Fig. 1; Lee et al., 2003). Location of the different protein domains within the hexamer is essential to understand their role in protein activity, allosteric communication within the hexamer, and thus the global mechanism of action of this disaggregase. "
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ABSTRACT: ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. Here, we summarize the use of sedimentation equilibrium approaches, complemented with sedimentation velocity and composition-gradient static light scattering measurements, to study the self-association properties of ClpB in dilute and crowded solutions. As the functional unit of ClpB is the hexamer, we study the effect of environmental factors, i.e., ionic strength and natural ligands, in the association equilibrium of ClpB as well as the role of the flexible N-terminal and M domains of the protein in the self-association process. The application of the nonideal sedimentation equilibrium technique to measure the effects of volume exclusion, reproducing in part the natural crowded conditions inside a cell, on the self-association and on the stability of the oligomeric species of the disaggregase will be described. Finally, the biochemical and physiological implications of these studies and future experimental challenges to eventually reconstitute minimal disaggregating machineries will be discussed.
Available from: Osmar Norberto de Souza
- "We used the homology modeling approach, implemented in the MODELLER [Sali and Blundell, 1993] 9v10 program, to build models of torsinAwt, delE302, delE303, F205I and R288Q based on the 3D structure of the ClpB second AAA+ domain of Thermus thermophilus (T. thermophilus) [Lee et al., 2003]. The protocol used to perform the molecular modeling experiments was: generation of 10 models, from which one model for each torsinA sequence was selected. "
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ABSTRACT: Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA+ protein family that localizes to the endoplasmic reticulum (ER) lumen and nuclear envelope (NE). This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients; R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy, and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA in order to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia. This article is protected by copyright. All rights reserved.
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