Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the PML-RBCC domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (T m ) reduction of 30°C compared to the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. Spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V and D219H mutants, in contrast to C213R, L217F and L218P mutations. Moreover, computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213 and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.