Calmodulin extracts the Ras family protein RalA from lipid bilayers by engagement with two membrane-targeting motifs

Abstract

Significance RalA and RalB are membrane-attached small GTPases that are crucial for tumorigenesis and uncontrolled growth in Ras-driven cancers. The C-terminal tails of several Ras and Rho family small G proteins, which are lipidated in vivo, bind to the calcium sensor calmodulin. 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 propose a mechanism for membrane removal of RalA, helping to shed light on the regulatory role that calmodulin plays in Ral signaling. This structure of a lipidated small G protein tail in complex with calmodulin may give clues about calmodulin interactions with other small G proteins, including K-Ras4B. RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown 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. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses 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.