Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.
Keywords: ANP32B, Acidic leucine-rich nuclear phosphoprotein family member B; BTFA, 3-bromo-1,1,1-trifluoroacetone; CD, Circular dichroism; CDK9, Cyclin-dependent kinase 9; CW, Continuous wave; Cc, Cytochrome c; Cytochrome c; DDR, DNA damage response; DEER, Double electron–electron resonance; DLS, Dynamic light scattering; DMEM, Dulbecco’s modified Eagle’s medium; DNA, Deoxyribonucleic acid; DTT, Dithiotreitol; Dmax, Maximum dimension; EDTA, Ethylenediamine tetraacetic acid; EGTA, Ethyleneglycol tetraacetic acid; EPR, Electron paramagnetic resonance; Encounter complex; FBS, Fetal bovine serum; GUI, Graphical user interface; HEK, Human embryonic kidney cells; HRP, Horseradish peroxidase; I2PP2A, Inhibitor 2 of the protein phosphatase 2A; I3PP2A, Inhibitor 3 of the protein phosphatase 2A; INTAC, Integrator-PP2A complex; IPTG, Isopropyl-β-D-1-thiogalactopyranoside; ITC, Isothermal titration calorimetry; Ip/Id, Intensity ratio of NMR resonances between paramagnetic and diamagnetic samples; LB, Luria-Bertani; MD, Molecular dynamics; MTS, (1-acetoxy-2,2,5,5-tetramethyl-δ-3-pyrroline-3-methyl) methanethiosulfonate; MTSL, (1-oxyl-2,2,5,5-tetramethyl- δ −3-pyrroline-3-methyl) methanethiosulfonate; MW, Molecular weight; Molecular dynamics; NAP1, Nucleosome assembly protein 1; NAPL, Nucleosome assembly protein L; NMA, Normal mode analysis; NMR, Nuclear magnetic resonance; NPT, Constant number, pressure and temperature; NVT, Constant number, volume and temperature; Nuclear magnetic resonance; OD600, Optical density measured at 600 nm; OPC, Optimal 3-charge, 4-point rigid water model; PCR, Polymerase chain reaction; PME, Particle mesh Ewald; PMSF, Phenylmethylsulfonyl fluoride; PP2A, Protein phosphatase 2A; PRE, Paramagnetic relaxation enhancement; PVDF, Polyvinylidene fluoride; Protein phosphatase 2A; RNA, Ribonucleic acid; RNApol II, RNA polymerase II; Rg, Radius of gyration; SAXS, Small-angle X-ray scattering; SC, Sample changer; SDS-PAGE, Sodium dodecylsulfate-polyacrylamide gel electrophoresis; SDSL, Site-directed spin labeling; SEC, Size-exclusion chromatography; SET/TAF-Iβ; SET/TAF-Iβ ΔC, SET/template-activating factor-Iβ construct lacking its C-terminal domain; SET/TAF-Iβ, SET/template-activating factor-Iβ; SPRi, Surface plasmon resonance imaging; TAF-Iα, Template-activating factor-Iα; TPBS, Tween 20-phosphate buffered saline; VPS75, Vacuolar protein sorting-associated protein 75; WT, Wild type; XRD, X-ray diffraction.
© 2022 The Author(s).