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Abstract

AAA proteins are part of the large superfamily of AAA+ proteins, which are ringshaped P loop NTPases, whose common function is unfolding macromolecules in an energy-dependant manner. AAA proteins usually consist of an N-terminal domain, and one or two ATPase domains named D1 and D2. ATPase domains are relatively conserved within the family of AAA proteins and they are also thought to mediate hexamerization. N-terminal domains are important for substrate recognition and binding and, in contrast to the ATPase domains, they vary in their folds. Based on published data and additional bioinformatic analysis of AAA proteins, we selected several different N-terminal domains from archaeal AAA proteins for functional and structural characterization. We also characterized proteins which share similar or related domains to the ones found in AAA proteins, making an important link between them. Heat and chemical aggregation assays of different substrate proteins were used to assay N-terminal domains, or full AAA proteins, for intrinsic chaperone activity. Protein structures were determined by crystallography or NMR spectroscopy. Results of this study indicate that the barrel-like N-terminal domains of AAA proteins originated from the similar nucleic acid binding domains. A change in the affinity for substrate, from nucleic acid to protein, may have occurred through different mechanisms in the evolution. In the case of double-psi barrels this has probably happened through the evolution of a simple beta-alpha-beta-motif found also in the RIFT and swapped hairpin barrels which are transcription factors, i.e. DNA binders. Structures of Mj0056 and SpoVT indicate that RIFT and swapped hairpin barrels have evolved further either by insertion of different structure elements (Mj0056) or by domain recruitment (SpoVT). Similarity between the PAN and ARC N-domains was found to be both in structure and function. Both domains comprise a coiled-coil followed by one or two OB folds. OB stands for oligosaccharide binding and indicates that also this domain originated from a DNA-binding fold. Structure of the ARC-Nc subdomain and a comprehensive analysis of chimeric constructs of the coiled coils and OB folds indicate that ARC and PAN N-domains have arisen through evolution by domain recruitment. Strict structural composition of the subdomains important in the chaperone function is maintained through the conserved PP-linker connecting the two subdomains. Structural and functional characterization of the AfAMA, a member of the novel group of AMA AAA proteins, showed that substrate binding function and chaperone activity of these proteins resides in its beta-clam like N-terminal domain. This domain can fulfill these functions independently, in contrast to the other homologous domains. We were able to show the importance of oligomerization for activity of these domains and that oligomerization is mediated by a small GYPL-motif found in a loop that presumably projects to the center of the hexamer. The N-terminal domain of AMA mediates hexamerization of the full protein independently from the AAA part of the protein and ATP utilization, which differs largely from other families of AAA proteins. Functional data on other beta-clam domains: VAT-Nc, Ph1500N and Hm-clam would indicate that these domains represent universal protein-protein interaction modules.