Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes

Dubey, Sudhisha; Mishra, Neha; Goswami, Nabajyoti; Siddiqui, Mohd Quadir; Varma, Ashok K.

doi:10.1016/j.ijbiomac.2022.11.022

Cited by 1 publication

(6 citation statements)

References 45 publications

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“…3A,B). The spectrum at 20 °C was characterized by a large negative change in ellipticity at λ = 208 and 222 nm, which is typical of a structured, predominantly α‐helical, wild‐type protein [24]. However, at 90 °C, significantly reduced ellipticity was observed with a slight change in the spectral pattern, except for the L217F and L218P mutants, which showed an insignificant difference in the spectral pattern.…”

Section: Resultsmentioning

confidence: 99%

“…The tendency of TRIM proteins to form oligomers is a significant obstacle to their structural analysis [28,29]. A study of PML-RBCC wild-type and mutants demonstrated that self-association is an inherent property of RBCC protein and does not require the presence of any other proteins in stoichiometric amounts [24]. The wild-type protein mainly exists as a tetramer, whereas the S214L and L217F mutations showed a predominant tetrameric state with a few oligomeric species.…”

Section: Discussionmentioning

confidence: 99%

“…After ATO treatment, wild-type, A216V, and D219H mutants showed positive ellipticity; however, insignificant differences were observed for C213R, L217F, and L218P. PML-RBCC contains three tryptophan residues; therefore, fluorescence spectra are sensitive to the environment [24]. Intrinsic fluorescence spectroscopy was used to monitor the alterations near tryptophan present in the wild-type and mutant proteins.…”

Section: Mutation C213r Is More Sensitive Towards Proteolysis Compare...mentioning

confidence: 99%

“…Mutations L211P, C212R, C213R, S214L, A216T, A216V, L217F, L218P, D219H, and S220G were generated through conventional PCR-based mutagenesis methods using PML-RBCC construct as a template and confirmed by DNA sequencing. Wild-type and recombinant mutant proteins were expressed and purified as reported earlier from our group [24].…”

Section: Site-directed Mutagenesis Expression and Purification Of Pml...mentioning

confidence: 99%

“…The PML-RBCC monomeric unit from the earlier reported tetrameric structure was used as a template for this study [24]. Furthermore, the residue of interest was identified and mutated as follows C213R, S214L, A216V, L217F, L218P, and D219H.…”

Section: Structure Modeling and Quality Assessmentmentioning

confidence: 99%

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

Dubey,

Mishra,

Shelke

et al. 2023

The FEBS Journal

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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 promyelocytic leukemia‐RING‐B‐box‐coiled‐coil (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 (Tm) reduction of 30 °C compared with 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. The spectroscopic analysis highlighted ATO‐induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Mutation C213r Is More Sensitive Towards Proteolysis Compare...mentioning

confidence: 99%

Section: Site-directed Mutagenesis Expression and Purification Of Pml...mentioning

confidence: 99%

Section: Structure Modeling and Quality Assessmentmentioning

confidence: 99%

See 3 more Smart Citations

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

Dubey,

Mishra,

Shelke

et al. 2023

The FEBS Journal

Self Cite

View full text Add to dashboard Cite

show abstract

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes

Cited by 1 publication

References 45 publications

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

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

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