Mapping of the domains involved in B-Myb-partner protein interactions and characterisation of their structural properties and features
In the work reported here, the minimal sites of interaction between B-Myb and its partner proteins p300 and cyclin D1 were mapped, and the structural properties and features of the interacting regions characterised in detail. A transactivation-associated central region of B-Myb was conclusively shown to bind to a region of p300 corresponding to the TAZ2, zinc ion binding domain. As is common for many transcriptional activation domains, the B-Myb transactivation region is intrinsically disordered in isolation but exhibits coupled folding upon binding p300. The specific B-Myb-p300 complex formed was found to adopt a 1:1 stoichiometry with a measured dissociation constant of less than ~3.0 X 10-8 M. An adjacent region of B-Myb containing part of exon 9a, was also found to interact with the preceeding ZZ region of p300, but substantially weaker than observed for theB-Myb-TAZ2 interaction. These findings clearly show that the conserved cysteine/histidine rich region 3 (C/H3) of p300 provides an extensive binding site for B-Myb.;In parallel work, a large central part of B-Myb containing both the transactivation region and exon 9a was found to show no binding to full-length cyclin D1. Together with previous work, this suggests that residues in the N-terminal DNA binding domain of B-Myb are also involved in cyclin D1 binding. Overall, this work suggests that the inhibition of cooperativity between B-Myb and p300 by cyclin D1 is not due to competition for a common binding site on B-Myb.