A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• The p53 transcription factor is crucial for maintaining genome integrity.
• p53 regulates genes involved in cell cycle arrest, apoptosis, and senescence.
• Mechanisms controlling p53 transcriptional activity, including post-translational modifications and binding partners, are complex and not fully understood.

Purpose of the Study:

• To develop a novel, non-invasive tool for manipulating endogenous p53.
• To investigate the potential of nanobodies targeting the p53 DNA-binding domain.
• To characterize the specificity and functional impact of these nanobodies on p53 activity.

Main Methods:

• Generation of nanobodies against the p53 DNA-binding domain.
• Co-crystal structure determination of p53 DNA-binding domain with nanobody Nb139.
• Conformation-specific antibody immunoprecipitations, glutaraldehyde crosslinking assays, and chromatin immunoprecipitation.
• Functional assays to assess the impact of nanobody binding on p53 target gene transactivation.

Main Results:

• Developed two distinct nanobodies, Nb3 and Nb139, targeting wild-type and mutant p53.
• Nb139 binds to p53 opposite the DNA-binding surface without disrupting its functional architecture.
• Nb139 binding effectively perturbs the transactivation of p53 target genes.
• Structural and functional data provide insights into p53 regulation.

Conclusions:

• Novel nanobodies offer a precise and non-invasive method to manipulate endogenous p53.
• Nb139 serves as a valuable tool for studying p53 function and regulation.
• The findings suggest that nanobody binding may affect transcriptional co-activator recruitment or post-transcriptional modifications.