Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics.
Apoptin is a 121 residue protein which forms large, soluble aggregates and possesses an exceptionally selectively cytotoxic action on cancer cells. In the accompanying paper, we described the design, production and initial characterization of an Apoptin truncated variant called H6-ApopΔProΔLeu. Whereas both the variant and wild type protein possess similar selective cytotoxicity against cancer cells following transfection, only the variant is cytotoxic when added externally. Remarkably, as observed by gel filtration chromatography and dynamic light scattering, H6-ApopΔProΔLeu lacks the tendency of wild type Apoptin to form large aggregates, which greatly facilitated the study of its biological properties. Here, we characterize the conformation and dynamics of H6-ApopΔProΔLeu. Using a battery of 2D, 3D and (4,2)D NMR spectra, the essentially complete 1H, 13C and 15N resonance assignments of H6-ApopΔProΔLeu were obtained. The analysis of these data shows that the variant is an intrinsically disordered protein, which lacks a preferred conformation. This conclusion is corroborated by a lack of protection against proteolytic cleavage and hydrogen/deuterium exchange. Moreover, the CD spectra are dominated by random coil contributions. Finally, 1H-15N NOE ratios are low, which indicates flexibility on the ps-ns time scale. Interestingly, H6-ApopΔProΔLeu's intrinsically disordered ensemble is not significantly altered by the redox state of its Cys residues or by Thr phosphorylation, which has been proposed to play a key role in Apoptin's selective cytotoxicity. These results serve to better comprehend Apoptin's remarkably selective anticancer action and provide a framework for the future design of improved Apoptin variants.
['Ruiz-Martínez|Santiago|S|', 'Pantoja-Uceda|David|D|', 'Castro|Jessica|J|', 'Vilanova|Maria|M|', 'Ribó|Marc|M|', 'Bruix|Marta|M|', 'Benito|Antoni|A|', 'Laurents|Douglas V|DV|']
D000970:Antineoplastic Agents D036022:Capsid Proteins D045744:Cell Line, Tumor D017727:Chicken anemia virus D003545:Cysteine D004354:Drug Screening Assays, Antitumor D019286:Endopeptidase K D006801:Humans D009682:Magnetic Resonance Spectroscopy D009369:Neoplasms D010766:Phosphorylation D011487:Protein Conformation D017510:Protein Folding D013050:Spectrometry, Fluorescence D013056:Spectrophotometry, Ultraviolet
Intrinsically disordered protein Phosphorylation Anti-cancer protein NMR spectroscopy