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

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.

RalA and RalB are members of the Ras superfamily of small GTPases, plasma membrane-associated molecular switches that regulate signal transduction affecting a plethora of cellular processes. Acting as one of the principal branches of the Ras signaling network, recruitment of a Ral-specific guanine exchange factor (RalGEF) promotes activation of RalA/B. Despite being less well studied than the MAPK and PI3K pathways, activation of RalGEFs is sufficient to induce Ras-driven transformation of human cells (1), and the inhibition of RalGEF disrupts colony formation in Ras-driven human cancer cell lines (2). It has also been reported previously that the RalGEF signaling pathway is crucial in the development of bone metastasis originating from pancreatic cancer in mice (3), and skin carcinoma mouse models deficient in RalGEF show decreased tumor size and number (4). Together, these findings indicate critical roles for both RalA and RalB in tumor formation and cancer progression, suggesting that it is important to expand our knowledge of their signaling roles and regulation.RalA and RalB share 82% sequence identity in their G-domains (guanine nucleotide-binding domain) and are almost identical structurally (5). Both proteins contain two switch regions, the conformations of which are sensitive to the bound nucleotide, allowing downstream effectors to select the active form of the protein. The effector binding sequences of RalA/B are identical, and it is therefore surprising that they display functional divergence in vivo, mediating distinct cellular effects in both normal cells and in cancer settings (6–10). Most of the sequence diversity between the Ral isoforms comes from the aptly named hypervariable region (HVR) located at the C terminus. HVRs are short, intrinsically disordered regions found in all Ras and Rho family proteins, which have recently come under scrutiny for their ability to interact with membranes to regulate and modify signaling output of the G domain [reviewed by Cornish et al. (11)]. Some of the HVRs may have a propensity for secondary structure formation, such as the K-Ras4B HVR, which is α-helical under certain circumstances (12).The C-terminal “CaaX box” motif (C = Cys, a = aliphatic, X = any residue) is the recognition sequence for isoprenylation of small GTPases, which facilitates their attachment to membranes. The C terminus of Ral proteins is recognized by GGTase-I (13), which adds a geranylgeranyl moiety to the Cys sidechain. The proteins are further processed by removal of the “aaX” motif and methylation of the new C-terminal carboxyl of the prenylcysteine (14). A secondary membrane attachment signal comes from positively charged Lys and Arg sidechains within the HVR, which interact with negatively charged phospholipid headgroups in the membrane bilayer. More than just a simple membrane anchor, the HVRs of RalA/B contain Ser residues that can be differentially phosphorylated in vivo. Ser194 of RalA is an Aurora kinase A target, phosphorylation of which has been shown to facilitate relocation to the mitochondrial membrane and binding to the effector RLIP76 (15).CaM (calmodulin) is a ubiquitous calcium sensor that regulates a multitude of partners. It is a small (16.7 kDa), pseudosymmetrical protein with two lobes (16), each comprising two EF-hand motifs. Upon calcium binding, the EF hands reorient to expose methionine-rich hydrophobic pockets that engage target proteins (17). The unusually high proportion of methionine residues, in conjunction with a flexible linker between the two lobes, confers extensive binding plasticity (18), allowing considerable sequence and structural diversity in CaM-interacting proteins. Binding often involves CaM wrapping around a positively charged helix in its target that contains hydrophobic anchors at defined positions in the sequence. These anchor residues dock into the hydrophobic pockets of the two CaM lobes. Alongside this canonical “wrap-around” mechanism, CaM also employs a variety of known noncanonical binding modes, which result in more extended conformations (19).CaM has been shown to interact with the HVRs of RalA and RalB in a Ca²⁺-dependent manner (19–21). It also binds the related small GTPase K-Ras4B in an interaction that involves burial of its C-terminal isoprenyl (farnesyl) group. Despite the interest in this interaction, there is no structure of K-Ras4B in complex with CaM. Although early data indicated that binding of K-Ras4B was nucleotide dependent, it is now thought that CaM binds to the prenylated C-terminal K-Ras4B HVR and that the nucleotide only controls accessibility of that region rather than binding directly to CaM (21). Biophysical data from one group indicated that two K-Ras4B molecules can bind to a single CaM, with the C-lobe of CaM binding around 10 times more tightly than the N-lobe (21). Another study used NMR titrations to show that the K-Ras4B protein is not necessary and that farnesyl compounds themselves are able to bind CaM (22). This was supported by a structure of a complex of CaM with farnesylated, methylated Cys, which binds exclusively to the C-lobe of CaM, in line with its higher affinity for that part of the CaM protein.The interaction between RalA and CaM has not been studied as extensively, and there are conflicts in the literature as to the number of CaM-binding sites on Ral (20, 23) and the cellular consequences of the interaction. One study found that the interaction with CaM stimulated the GTPase “off-switch” of RalA (24), but another report showed that CaM binding caused RalA activation (20). Previous work has alluded to the importance of the prenyl anchor in the interaction (25), although there is a lack of biophysical data to corroborate and explain this observation. An in-depth structural and biophysical analysis of the RalA–CaM complex would allow an assessment of the potential functional consequences of this interaction. In this investigation, using maleimide-conjugated prenyl mimics, we sought to elucidate the molecular basis for the interaction between RalA and CaM to better understand its role in vivo. We establish that the binding motif for CaM is within the RalA-HVR and demonstrate the importance of the prenyl anchor for high affinity binding. We have solved the first structure of CaM in complex with a lipid-modified HVR, which shows that the N-lobe of CaM encases the prenyl group whereas the C-lobe interacts with key hydrophobic residues of the HVR. Furthermore, we show that CaM is able to extract RalA from the surface of a lipid membrane and propose a stepwise temporal order for the mechanism. Other small GTPases suspected of interaction with CaM, such as RalB, Rac1, Cdc42 (26), and Rap1A (27), have features in common with RalA, including a polybasic motif and prenyl moieties at their C termini. The results presented here may therefore provide a framework to understand the interaction of CaM with these proteins.