Characterization of the ribosome-associated complex RAC from S. cerevisiae

Abstract

Zuotin and Ssz from yeast are members of the conserved Hsp40 and Hsp70 family of molecular chaperones, respectively, and are thought to be involved in the folding of newly synthesized proteins. Hsp70s use an ATP-controlled cycle for substrate binding and release which is regulated by their Hsp40 co-chaperones. However, in contrast to canonical Hsp70/40 members, Zuotin and Ssz form an unusually stable heterodimer termed ribosome-associated complex (RAC), which is bound to the ribosome. Thus RAC acts as a co-chaperone for another ribosome-bound Hsp70, Ssb. The aim of this thesis was to elucidate the unusual pairing of RAC and the influence of ATP on the complex by investigating the conformational changes and dynamics in solution. These conformational studies were performed using amide hydrogen exchange (HX) combined with high resolution mass pectrometry (MS). Additional analyses were conducted using mutational analyses, fluorescence measurements and electron paramagnetic resonance (EPR) measurements. HX-MS experiments with recombinant, purified S. cerevisiae Ssz, revealed specific ATP-induced conformational changes in Ssz individually and in complex. Upon ATP binding the nucleotide binding domain (NBD) was stabilized, resulting in a more compact structure. However, in contrast to canonical Hsp70s no influence of ATP binding on the substrate binding domain (SBD) was observed. Complex formation did not influence the nucleotide binding domain, but decreased the conformational dynamics within the SBD, indicating the engagement of the SBD in complex formation. Biochemical binding analysis of the individual domains NBD and SBD, respectively, revealed an interaction of the NBD of Ssz with the N-terminal domain of its complex partner Zuo. This indicates an involvement of both domains (NBD and SBD of Ssz) in complex formation of RAC. HX-MS experiments with recombinant, purified S. cerevisiae Zuo, revealed an extremely flexible region at the beginning of the N-terminal region of Zuo (aa 1 - 79). Upon complex formation this structure was conformationally stabilized, leaving only a short linker-like-region (aa 53 - 79) unstructured. Deletion of the highly flexible Nterminus of Zuo abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz’s function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuo and their mutual stabilization are the major structural determinants for RAC assembly. Moreover, HX-MS experiments revealed dynamic changes in the J-domain of Zuo upon complex formation that might be crucial for RAC’s co-chaperone function. Further EPR analysis using cysteine mutants of Zuo indicated that the conformational changes are not restricted to the interacting HPD motif itself but rather to the whole J-domain. The results obtained in this work allowed to develop a model presenting a novel mechanism for converting Zuo and Ssz chaperones into a functionally active heterodimer.