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Abstract

AAA proteins are part of the large superfamily of AAA+ pro- teins, ring-shaped P loop NTPases, which display their function by 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 the hexamerization of AAA proteins. N-terminal domains are important for substrate recog- nition and binding and, in contrast to the ATPase domains, they vary in their folds. Based on published data and additional bioin- formatic analysis of AAA proteins, we selected several different N-terminal domains from archaeal AAA proteins for functional and structural characterization. All selected domains were expressed as recombinant proteins in Escherichia coli. Guided by prior knowledge that AAA proteins have a protein unfolding function, we used heat and chemical aggregation assays of differ- ent substrate proteins to assay N-terminal domains, or full AAA proteins, for possible chaperone activity. All constructs showed activity in inhibition of aggregation of protein substrates. Sur- prisingly, we found that the N-terminal domains could them- selves play a role in the hexamerization of AAA proteins, a role previously assigned to the ATPase domains. The results of our study indicate that AAA ATPase N-terminal domains of AAA proteins containing different unrelated structures share a com- mon function, namely intrinsic chaperone activity.