Binding to retinoblastoma pocket domain does not alter the inter-domain flexibility of the J domain of SV40 large T antigen

Abstract

Simian Virus 40 uses the large T antigen (Tag) to bind and inactivate retinoblastoma tumor suppressor proteins (Rb), which can result in cellular transformation. Tag is a modular protein with four domains connected by flexible linkers. The N-terminal J domain of Tag is necessary for Rb inactivation. Binding of Rb is mediated by an LXCXE consensus motif immediately C-terminal to the J domain. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) were used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N(260)) extending from the J domain through the origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the domains in N(260). Binding of pRb to a construct containing the LXCXE motif and the J domain revealed weak interactions between pRb and the J domain. Analysis of the complex of pRb and N(260) indicated that the OBD is not involved and retains its dynamic independence from the remainder of Tag. These results support a 'chaperone' model in which the J domain of Tag changes its orientation as it acts upon different protein complexes.