Spatial and functional interrelationship of a heterotetramer Survivin-DNA-PKcs complex in the repair of DNA double-strand breaks

Abstract

Survivin was discovered as a member of the Inhibitor of Apoptosis Protein (IAP) family showing high expression in almost all human cancers. Although primarily considered as a protein implicated apoptosis and cell cycle/mitotic spindle checkpoint regulation, Survivin is now recognized as a nodal factor involved in a multitude of cellular circuits. By this, Survivin covers a radiation resistance factor in a variety of cancer entities and enhances tumor cell survival upon radiation exposure by impacting DNA double-strand break (DNA DSB) repair. Following irradiation, nuclear accumulation of Survivin was mechanistically been linked to the activity of the DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key component of DNA DSB repair pathway non-homologous end joining (NHEJ). In this study, we aimed to unravel the determinants of the Survivin-DNA-PKcs interrelationship on a molecular level by computational investigations of the regions of interaction and biochemical approaches. 3D crystallographic structures of Survivin and catalytic PI3K domain of DNA-PKcs were virtually docked using advanced global docking algorithms, simulated by molecular dynamics, and were evaluated according to binding free energies (ΔG) and the spatial accessibility/physical proximity. Next, multiple residues derived from these analyses were mutated, and the functional consequences of the mutagenesis were assayed by flow cytometry-based Forster resonance energy transfer (FACS-FRET) and co-immunoprecipitation (co-IP) experiments. Radiation survival and DNA damage repair capacity were assayed by 3D colony formation assays and DNA foci analysis (γH2AX/53BP1), respectively. The effects of the Survivin-DNA-PKcs interrelationship were further analyzed by in vitro DNA-PKcs kinase activity assays and Liquid Chromatography-Mass Spectrometry (LC-MS2/3)-based multi-proteomic techniques. Finally, a virtual drug screening approach was employed in search for novel small-molecule radiosensitizers targeting the Survivin-DNA-PKcs interaction. Molecular docking and advanced in silico analyses uncovered residues serine(S)20 and tryptophan(W)67 located in the baculovirus inhibitor of apoptosis protein repeat (BIR) domain of dimerized Survivin to interact with the PI3K domain of DNA-PKcs. Mutagenesis of these residues significantly decreased the interaction compared to wild-type (wt) Survivin, was correlated with an increased radiosensitivity of colorectal cancer cells and a hampered DNA repair capacity, measured by γH2AX/53BP1 foci analysis, after knockdown of endogenous Survivin. By contrast, overexpression of wt Survivin rescued radiation survival and DNA repair. In addition, advanced molecular docking and dynamics simulation analyses revealed a heterotetramer model, where Survivin binds to the surface of pre-existing DNA-PKcs dimer. Moreover, by investigating the effects of Survivin on DNA-PKcs’ downstream regulatory functions, differentially abundant phosphopeptides and proteins were identified for multiple pathways, predominantly for DNA damage/repair. Binding of Survivin to a pre-existing DNA-PKcs dimer was lead to a conformational change on the PI3K domain and resulted in a differential change in substrate specificity. Particularly, the previously little-known DNA-PKcs’ S/T-Hydr (hydrophobic residues: G, A, V, L, I, P, F, M, W) motif substrates including the FOXO3 S253 phosphosite displayed high conservation within the detected phosphosites. Further, proteomics analyses indicated that the Survivin-DNA-PKcs interrelationship not only displays post-translational but also protein expression-level regulatory properties. Ultimately, the virtual drug screening approach uncovered small-molecule compounds having strong binding affinity to S20, and W67 residues and consequently might show promise for the development of future radiation sensitizing therapeutic approaches. In summary, in this study, we identified specific residues of Survivin involved in the interaction with the PI3K domain of DNA-PKcs by implementing in vivo live cell protein interaction quantification and in silico structure-based molecular docking technologies. Besides that, findings on radiosensitivity, DNA foci formation, kinase activity, and phosphoproteomics and proteomics analyses further strengthen the notion that Survivin is a fine-tuning regulator of DNA DSB repair and impacts on substrate specificity by fostering the S/T-Hydr motif phosphorylation. Large-scale proteomics and phosphoproteomics studies further discovered novel candidate proteins and phosphosites, enlightening the underlying mechanistic relation between Survivin and DNA-PKcs in response to irradiation and may pave the way to novel Survivin-related cancer and DNA damage response marker discoveries.