Thermodynamic and Kinetic Characterization of MST1 and Rassf5 conserved Sav/Rassf/Hpo (SARAH) Domain Interactions

Abstract

The molecular switch Ras exhibits its biological function- control of growth, differentiation and apoptosis through the interaction with a multitude of different effectors. It is apparent that growth-inhibitory properties of Ras are mediated via noncatalitic polypeptides of Rassf (Ras Association Domain Family). Tumour suppressor Rassf5 (also termed Nore1) binds directly to active Ras via the Ras Binding Domain (RBD). It is also known to form self-associated complex as well as heterodimers with the proapoptotic serine/threonine Mammalian Sterile 20-like kinase (MST1), the human ortholog for Hippo (Hpo), through their common conserved C-terminal Sav/Rassf/Hpo (SARAH) domains [1, 2]. This unique interaction motif connects the proteins involved in the recent discovered pathway mediated by proteins of the MST family, which promotes apoptosis and restricts cell proliferation [3-6].For a better understanding of MST1 and Nore1 homo- and hetero- interactions via the SARAH domains, we have investigated the thermodynamics and kinetics of association/dissociation as well as the unfolding mechanism of this domain by use of different biophysical and biochemical methods, such as Differential Scanning Calorimetry (DSC), size-exclusion chromatography, artificial chemical cross-linking, Isothermal Titration Calorimetry (ITC), Circular Dichroism Spectroscopy (CD). MST1 and Nore1 SARAH domains are shown to form not only homodimers, but also higher oligomers. Nevertheless, the heterodimers rather than homodimers are preferentially formed. Finally, we propose a possible mechanism for the thermal unfolding of MST1 and Nore1 SARAH homo- and heterocomplexes.