Proteins must fold correctly in order to attain biological function. Concurrently, protein misfolding and aggregation are primary contributors to many human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and transmissible spongiform encephalopathy (TSE), better known as the human form of "Mad Cow Disease". Molecular chaperones, such as Hsp60 (GroEL), Hsp70 (DnaK) and Hsp90 (HtpG), assist protein folding by either promoting the "forward" folding or preventing the aggregation of proteins. However, once aggregates have formed, these molecular chaperones cannot facilitate protein disaggregation.
Hsp104 (ClpB) is a 600 kDa ATP-dependent molecular machine that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. My lab is interested in understanding how ClpB converts the energy derived from ATP binding and/or hydrolysis into mechanical work in order to disaggregate previously aggregated proteins. To this end, we have solved the 3-Å resolution crystal structure of ClpB (PDB: 1QVR) using X-ray crystallography, and the structure of the functional ClpB assembly using electron cryo-microscopy and single-particle reconstruction techniques (Lee et al. Cell 2003).
Figure 1: Mechanistic Model for Protein Disaggregation by ClpB. (1) Disaggregation of high molecular weight aggregates which bind to the outside of the ClpB hexamer. We propose that ClpB recognizes only large aggregates that can bind simultaneously to motif-1 and motif-2 of the M-domain of neighboring ClpB subunits. The red arrows indicate the concerted and opposite motion of motif-1 and motif-2 in adjacent subunits, which could generate the mechanical force to pull apart large aggregates. (2) Resolubilization of medium size aggregates or misfolded polypeptides. Following protein disaggregation, the smaller aggregates are either recognized by the DnaK chaperone system directly or enter the cavity of the ClpB hexamer, where they are resolubilized perhaps by (2a) a "capture-and- release" or (2b) a translocation mechanism. (3) Refolding of small aggregates or polypeptides by the DnaK chaperone system (Figure adapted from Lee et al., 2003).
To investigate the structure-function relationship of ClpB, we have engineered a ClpB variant (BAP) by replacing a helix-loop-helix motif in ClpB with the analogous motif of ClpA, which contains the conserved IGF/L tripeptide motif required for ClpP binding. Unlike ClpB, BAP can associate with the ClpP protease in a stable, ATP-dependent manner. While ClpB and BAP share the ability to disaggregate proteins, in the presence of ClpP, BAP (but not ClpB) functions as a novel disaggregating-degrading machine. Using BAP, we demonstrated that substrates must translocate through the central pore of the ClpB hexamer and, perhaps more importantly, that thermotolerance requires the refolding of aggregated proteins, i.e. it is not the aggregate itself that causes cell death (Weibezahn et al. Cell 2004).
While the structure-function relationship is beginning to be understood, the ATP-driven conformational changes that occur in the ClpB hexamer are unknown. Moreover, it remains unclear how the nucleotide-driven conformational changes are coupled to substrate recognition and binding. To provide the structural basis for substrate binding, we determined the single-particle, cryo-EM reconstruction of the ATP-activated state of ClpB (EMDB: 1244) by examining the structure of a ClpB mutant that binds but does not hydrolyze ATP (Trap-ATP). In addition, we also obtained reconstructions of ClpB wild-type in the AMPPNP (EMDB: 1243), ADP (EMDB: 1242), and nucleotide-free states (EMDB: 1241). Each structure represents a snapshot of ClpB in a different nucleotide-state, and together the structures provide a molecular understanding of the ATP-driven conformational changes as they may occur in solution. By fitting the crystal structure of a ClpB monomer (PDB: 1QVR) into our cryo-EM reconstruction of the Trap-ATP and ClpB-AMPPNP hexamers, we showed that motif 2 of the ClpB M-domain is positioned between the D1-large domains of neighboring subunits (Haslberger et al., Mol. Cell 2007) and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. Furthermore, we demonstrated using fluorescence polarization that high-affinity substrate binding to ClpB requires ATP and, to our surprise, cannot be substituted with AMPPNP. Comparison of the Trap-ATP and ClpB-AMPPNP hexamer structures revealed that, in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring and threads them through the ClpB hexamer in an ATP hydrolysis-driven step (Lee et al. Mol. Cell 2007).
See BCM Press Release "Correcting misfolded proteins" by G. Gutierrez.