title: Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics.
abstract: 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 complete1H,13C and15N 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.
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, and Institut d'Investigació Biomedica de Girona Josep Trueta (IdIBGi), c/ Maria Aurèlia Campmany 40, 17003, Girona, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Ruiz-Martínez
authors.affiliation: Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, c/ Serrano 119, 28006, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pantoja-Uceda
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, and Institut d'Investigació Biomedica de Girona Josep Trueta (IdIBGi), c/ Maria Aurèlia Campmany 40, 17003, Girona, Spain.
authors.forename: Jessica
authors.initials: J
authors.lastname: Castro
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, and Institut d'Investigació Biomedica de Girona Josep Trueta (IdIBGi), c/ Maria Aurèlia Campmany 40, 17003, Girona, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, and Institut d'Investigació Biomedica de Girona Josep Trueta (IdIBGi), c/ Maria Aurèlia Campmany 40, 17003, Girona, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.affiliation: Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, c/ Serrano 119, 28006, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, and Institut d'Investigació Biomedica de Girona Josep Trueta (IdIBGi), c/ Maria Aurèlia Campmany 40, 17003, Girona, Spain. Electronic address: antoni.benito@udg.edu.
authors.forename: Antoni
authors.initials: A
authors.lastname: Benito
authors.affiliation: Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, c/ Serrano 119, 28006, Madrid, Spain. Electronic address: dlaurents@iqfr.csic.es.
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry*; D045744:Cell Line, Tumor; D017727:Chicken anemia virus; D003545:Cysteine / Q000737:chemistry; D004354:Drug Screening Assays, Antitumor; D019286:Endopeptidase K / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009369:Neoplasms / Q000473:pathology* / Q000628:therapy*; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000970:Antineoplastic Agents; D036022:Capsid Proteins; C091717:VP3 protein, Chicken anemia virus; D019286:Endopeptidase K; D003545:Cysteine
keywords: Anti-cancer protein; Intrinsically disordered protein; NMR spectroscopy; Phosphorylation