Biochemical and structural analysis of SV40 Large T antigen: insights into chaperone mediated inactivation of retinoblastoma tumor suppressor protein

Abstract

Simian Virus 40 uses its Large T antigen protein to bind and inactivate retinoblastoma tumor suppressor proteins, yielding cell transformation. T antigen is a modular protein with four domains connected by linkers. The N-terminal domain, the J domain, classifies T antigen as a chaperone protein and is necessary for the inactivation of Rb. Physical interaction with Rb is mediated primarily by an LXCXE consensus motif immediately C-terminal to the J domain. An approach integrating nuclear magnetic resonance (NMR) spectroscopy and small angle x-ray scattering (SAXS) was used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N260) that extends from the J domain to the adjacent origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the two domains in N260. Rb bound to a construct containing the LXCXE motif and the J domain confirmed that weak interactions exist between Rb and the J domain. Analysis of the interaction of Rb with N260 indicated that the OBD is not involved in binding Rb and that the J domain retains dynamic independence from the remainder of T antigen. These results support a T antigen ‘chaperone’ model in which the J domain orientation changes as it acts upon protein complexes to promote cell transformation.

The ubiquitin ligase Cullin 7 (Cul7) has been implicated in chaperone-mediated inactivation of Rb. It is known that Cul7 binds to T antigen via residues within the J domain. However, the Cul7 domain involved in this interaction is unknown. Yeast two hybrid screens in the Ellen Fanning laboratory suggest that the CPH domain of Cul7 bound to T antigen. NMR chemical shift perturbation experiments confirmed the interaction between the J domain of Tag and CPH domain of Cul7. These data identified specific CPH domain residues involved in binding to the Tag J domain, which map to a specific binding surface. Our results set the stage for mutational analysis to establish the functional role of the Tag-Cul7 interaction.