Structural and functional analysis of cancer-associated missense variants in the retinoblastoma protein (Rb) pocket domain

The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors that bind via an LxCxE motif. The pocket domain is the most highly-conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rbs pocket domain, we used fluorescence polarization and differential scanning fluorimetry to quantify changes, caused by 75 cancer-associated missense variants, to E2F transactivation domain (E2FTD) binding, LxCxE binding, and changes to the thermostability of the protein. We find that 43% of the missense variants we tested reduce Rb-E2FTD binding. Many of these variants are not located at the E2F binding site, yet they destabilize the fold of the protein and show temperature-sensitive binding effects. We also find that 21% of tested mutations reduce LxCxE binding, and several mutations selectively disrupt either E2FTD or LxCxE binding. Protein X-ray crystallography of four missense variants reveals how different mutations destabilize the protein fold and inhibit E2FTD or LxCxE binding. Taken together, this work provides the first understanding of the multiple ways through which stability, structure and function of Rbs pocket domain is altered by a large number of missense mutations seen in cancer.