title: A nanobody modulates the p53 transcriptional program without perturbing its functional architecture.
abstract: The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds 'structural' mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations.
authors.affiliation: Nanobody Lab, Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Jonas
authors.initials: J
authors.lastname: Bethuyne
authors.affiliation: Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: De Gieter
authors.affiliation: Nanobody Lab, Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Zwaenepoel
authors.affiliation: Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors.forename: Abel
authors.initials: A
authors.lastname: Garcia-Pino
authors.affiliation: Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
authors.forename: Kaat
authors.initials: K
authors.lastname: Durinck
authors.affiliation: Nanobody Lab, Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Adriaan
authors.initials: A
authors.lastname: Verhelle
authors.affiliation: Nanobody Service Facility, Vlaams Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Hassanzadeh-Ghassabeh
authors.affiliation: Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.affiliation: Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors.forename: Remy
authors.initials: R
authors.lastname: Loris
authors.affiliation: Nanobody Lab, Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium jan.gettemans@ugent.be.
authors.forename: Jan
authors.initials: J
authors.lastname: Gettemans
mesh_terms: D002460:Cell Line; D006801:Humans; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000276:immunology / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*
chemical_list: D061905:Single-Domain Antibodies; D016159:Tumor Suppressor Protein p53