Surface charge of Merkel cell polyomavirus small T antigen determines cell transformation through allosteric FBW7 WD40 domain targeting

Abstract

AbstractMerkel cell polyomavirus (MCV) small T (sT) is the main oncoprotein in Merkel cell carcinoma (MCC) development. A unique domain of sT, LT stabilization domain (LSD), has been reported to bind and inactivate multiple SCF (Skp1-Cullin-F-box) E3 ligases. These interactions impede the turnover of MCV large T (LT) antigen and cellular oncoproteins such as c-Myc and cyclin E, thereby promoting viral replication and cell transformation. However, it is currently unclear how this LSD region contributes to multiple transforming activities of sT. Structural docking simulation of sT and F-box and WD repeat domain-containing 7 (FBW7) revealed a novel allosteric interaction between sT and FBW7 WD40 domain. This model is supported by experimental evidence confirming that charge engineering in the LSD alters sT-WD40 binding. Specifically, loss of net positive charge in the LSD prevents sT-FBW7 binding by abrogating the electrostatic interaction, thus impeding inhibition of FBW7 by sT. Furthermore, positively charged mutations in the LSD significantly restored the sT function and its ability to transform rodent fibroblast cells. We infer that the surface charge of sT is a major determinant for targeting E3 ligases, which leads to sT-induced cell transformation, an observation that could be used to develop targeted therapeutics for MCC.