Structural dissection of three transition states along the folding pathway of PDZ6 from PDZD2

: The cooperative nature of protein folding limits the experimental dissection of the reaction mechanism. PDZ domains, with conserved folds, numerous homologs, and accessible folding intermediates, offer an ideal model to study folding pathways. Here, we present a detailed structural and kinetic characterization of the folding pathway of PDZ6 from PDZD2. Using kinetic folding experiments under different salt conditions combined with φ-value analysis, we revealed a complex energy landscape for PDZ6, featuring three distinct transition states (TS1-TS3) and the progressive acquisition of native-like structure along the reaction coordinate. Taking advantage of the large number of homologous PDZ domains, we compared φ-values at conserved structural positions with those previous obtained for PDZ3 of PSD-95 and PDZ2 of PTP-BL. This analysis revealed a shared, conserved folding mechanism among PDZ domains, in which the central β-strands act as nucleation cores for folding. Overall, this work provides the first structural dissection of the three transition states governing PDZ6 folding and highlights a conserved, hierarchical folding mechanism among PDZ domains. These findings expand our understanding of PDZ folding principles and may inform studies on their functional modulation and evolutionary adaptation.