Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a

Abstract

We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 plays a critical role in the cell cycle by inhibiting cyclin-dependent kinases CDK4 and CDK6, and it is inactivated in sporadic and familial cancers. The majority of the p16 missense mutations cause loss of function by destabilizing the proteins structure. We show that the nanobodies bind p16 with nanomolar affinities and restore the stability of a range of different cancer-associated p16 mutations located at sites throughout the protein. The nanobodies also bind and stabilize p16 in a cellular setting. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. These findings indicate that nanobodies could be used as pharmacological chaperones to determine the consequences of restoring the function of p16 in the cell. HighlightsO_LIDescribes a nanobody (single-domain antibody) capable of binding tumor suppressor protein p16 C_LIO_LINanobody binding can stabilise wild-type and cancer-associated p16 mutants C_LIO_LINanobody-p16 crystal structure reveals why nanobody binding is compatible with p16-CDK6 interaction C_LI