EXPLORING THE FOLDING PATHWAYS OF ANNEXIN-I, A MULTIDOMAIN PROTEIN - II - HIERARCHY IN DOMAIN FOLDING PROPENSITIES MAY GOVERN THE FOLDING PROCESS

In the context of exploring the relationship between sequence and fold ing pathways, the multi-domain proteins of the annexin family constitu te very attractive models. They are constituted of four similar to 70- residue domains, named D1 to D4, with identical topologies but only li mited sequence homology of approximately 30%. The domains are organize d in a pseudochiral circular arrangement. Here, we report on the foldi ng propensity of the D1 domain of annexin I obtained from overexpressi on in Escherichia coli. Unlike the D2 domain, which is only partially folded, the isolated D1 domain exhibits autonomous refolding in pure a queous solution. Similarly, the D3 domain and D2-D3 module were obtain ed from expression in E. coli but were found to be largely unfolded. N o conclusion could be drawn for the D4 domain because it was not possi ble to extract it from the bacterial inclusion bodies. The data allow us to propose a plausible scenario for the annexin I folding. This wor king model states that firstly the D1 domain folds, and the D2 and D3 domains remain partly unfolded, facilitating the docking of the D4 dom ain to the D1 domain. In a second step, the D1 and D4 domains dock, an d D4 may fold if already not folded. The final step starts with the st abilization of the D1-D4 module. This stabilization is crucial for all owing the nonnative local interactions inside the still partially unfo lded D2 domain to switch to the native long-range interactions involvi ng D4. This switch allows the complete folding of D2 and D3. The model proposes a sequential and hierarchical process for the folding of ann exin I and emphasizes the role of both native framework and non-native structures in the process. (C) 1998 Academic Press.