Calmodulin extracts the ras family protein rala from lipid bilayers by engagement with two membrane-targeting motifs.

Abstract

RalA is a membrane-attached small GTPase being crucial for tumorigenesis and uncontrolled growth in Ras-driven cancers. Its function as molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. It has been shown before that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. In this work, using a range of structural and biophysical methods (nuclear magnetic resonance, isothermal titration calorimetry and surface plasmon resonance), we have solved the structure of calmodulin in complex with the lipidated C-terminal tail of RalA and show that complex formation leads to RalA being removed from the lipid bilayer. We found that CaM interacts exclusively with the C-terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Furthermore, we show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B aiding to find new ways of targeting GTPases in cancer. (1) ((1) DOI: https://doi.org/10.1073/pnas.2104219118).