MECHANISM OF ACTION OF THE HSP100 CHAPERONE CLPA

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DESCRIPTION (applicant's description): The objective of the proposed studies is to provide a mechanistic understanding of ATP-dependent unfolding and proteolysis mediated by HSP100 chaperone ring structures in association with cylindrical proteases, resembling the action of 19S "cap" assemblies in directing proteins for degradation by the eukaryotic 20S proteasome cylinder. In particular, we are studying the mechanism by which the bacterial HSP100 chaperone, CIpA, a hexameric ring with two ATP binding domains in each of its 84 kDa subunits, mediates ATP dependent unfolding of protein substrates and commits them to translocation into and degradation by the coaxially bound cognate protease, CIpP, a stacked double ring tetradecamer of identical 23 kDa serine protease subunits. Translocation of several model substrates will be studied using fluorescence dynamics measurements, assessing whether the unfolded proteins are directionally translocated into ClpP. The hypothesis that proteolysis does not commence until substrate translocation is completed will be tested using fusion proteins. The site of substrate binding on ClpA will be determined by EM analysis of gold-tagged substrate proteins bound to CIpAP complexes. The dynamic ATP-directed movements of ClpA in directing unfolding and translocation will also be studied, using rapid-freezing and cryoEM examination of CIpAP complexes in various nucleotide states. Structure-function analyses will be carried out, employing random and site-directed mutants of CIpA, both in vivo and in vitro, to study function, and crystallographic studies of various forms of CIpA to obtain high-resolution structural information.