ATP binding to nucleotide binding domain (NBD)1 of the ClpB chaperone induces motion of the long coiled-coil, stabilizes the hexamer, and activates NBD2.
ABSTRACT The molecular chaperone ClpB can rescue the heat-damaged proteins from an aggregated state in cooperation with other chaperones. It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1. In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-A-long coiled-coil that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the accessibility of a labeling reagent, fluorescence change of a labeled dye, and cross-linking between the coiled-coil and NBD1 by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.
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ABSTRACT: The MecA-ClpC complex is a bacterial type II AAA+ molecular machine, responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA+ proteins are long conceived, the detailed mechanistic model remains elusive. With cryo-electron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA+ rings, and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of fashions, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two NBD rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA+ rings, and suggest that concerted actions of two AAA+ rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase, and especially, lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA+ hexamers.Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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ABSTRACT: Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, regions that are capable to include intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA+ proteins, because in addition to the classic AAA+ module they contain a part of another AAA+ module, namely the alpha-helical domain including a coiled-coil region, which is similar to the alpha-helical domain of the AAA(+)-1 module of the chaperone-disagregases ClpB/Hsp104.Bioorganicheskaia khimiia 39(3):303-19.
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ABSTRACT: Protein quality control within the cell requires the interplay of many molecular chaperones and proteases. When this quality control system is disrupted, polypeptides follow pathways leading to misfolding, inactivity and aggregation. Among the repertoire of molecular chaperones are remarkable proteins that forcibly untangle protein aggregates, called disaggregases. Structural and biochemical studies have led to new insights into how these proteins collaborate with co-chaperones and utilize ATP to power protein disaggregation. Understanding how energy-dependent protein disaggregating machines function is universally important and clinically relevant, as protein aggregation is linked to medical conditions such as Alzheimer's disease, Parkinson's disease, amyloidosis and prion diseases.Nature Reviews Molecular Cell Biology 10/2013; 14(10):617-29. · 37.16 Impact Factor