bioRxiv Science⌕ Search

Biology subjects

Muller, G.

Publications and source records attributed to Muller, G..

2 recordsLinked to original sources
bioRxiv2022

B-Myb association with DNA is mediated by its negative regulatory domain and Cdk phosphorylation

B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb binding site (MBS) sequences using their DNA binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb dependent promoters. The structural mechanisms underlying autoinhibition and activation have been poorly characterized. We determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits DBD binding to the MBS DNA sequence. We demonstrate that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provides insight into how B-Myb functions as a site-specific transcription factor.

biochemistry↗
bioRxiv2022

Structural basis for tunable affinity and specificity of LxCxEdependentprotein interactions with the retinoblastoma protein family

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle. Functional inactivation of Rb family proteins leads to loss of cell-cycle control and promotes genome instability and proliferation, which are hallmarks of cancer. Rb proteins share a structural domain, known as the "pocket domain", which mediates association with a large number of cellular proteins. A cleft in the pocket domain binds an LxCxE sequence motif in these proteins, many of which function with Rb proteins to co-regulate transcription during quiescence and G1. Proteins from oncogenic DNA viruses also bind this cleft to inactivate Rb family proteins, and the E7 protein from the human papillomavirus has been used as the primary model for understanding LxCxE motif interactions. Evidence presented previously and here demonstrates that the E7 sequence binds tighter and competes with cellular proteins for the LxCxE site, but the molecular basis for the relatively weak affinities of the cellular proteins has been poorly understood. Here, we present binding data and a series of crystal structures of the p107 pocket domain in complex with LxCxE peptides derived from the transcriptional co-repressor proteins HDAC1, ARI4DA, and EID1. The structures reveal determinants of protein binding beyond the L-C-E core that include the N and C-terminal sides of the LxCxE motif and the x (wild card) residues. Our results explain how differences in these interactions outside the L-C-E core result in weaker affinities of pocket protein for cellular LxCxE proteins compared to E7 and result in differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their myriad of cell partners and lays the foundation for structure-based design of peptides that inhibit oncogenic viral protein interactions.

biochemistry↗