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

Abstract

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.

Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB

“…This is consistent with the slightly weaker binding of lipidated RalB to CaM compared with that of lipidated RalA. [10] Given that we know that HVR engagement is necessary for membrane extraction, it is therefore likely that although CaM can extract RalA from membranes in vivo it cannot do the same for RalB. It has previously been reported that CaM interacts with both RalA and RalB, [11,12] but in these experiments solubilised membrane extracts were used, which exposes the lipid moiety.…”

“…When C-lobe interaction with the HVR is blocked, by the introduction of an RalA L195A/I199A double mutation, extraction from the membrane is ablated. [10] NMR experiments were used to compare binding of non-lipidated peptides corresponding to the RalA and RalB HVR [10] (Figure 1A). The peptides were titrated into 15 N labelled CaM, and HSQC experiments were recorded at each titration point.…”

“…(A) RalA and RalB HVR peptides were titrated into 15 N-labelled CaM and HSQC experiments performed as reported previously. [10] The RalA titration is shown on the left while the RalB titration is on the right. In both spectra the titration points are coloured as follows: 50 μM CaM alone is red, addition of 0.25 (orange), 0.5 (yellow), 1 (green), 1.…”

“…Binding to CaM results in the extraction of RalA from the membrane, due to sequestration of the lipid moiety at the C‐terminus of RalA within a cage‐like structure formed by the N‐terminal lobe of CaM. [ 10 ] In contrast, the C‐terminal lobe of CaM induces the formation of a short helix in the HVR, with two hydrophobic RalA residues, Leu195 and Ile199, projected into a hydrophobic cleft on the C‐lobe. This capture of the HVR at the membrane is a key first step in the extraction of the C‐terminal lipid moiety from the membrane, which leads to solubilisation of RalA.…”

“…When C‐lobe interaction with the HVR is blocked, by the introduction of an RalA L195A/I199A double mutation, extraction from the membrane is ablated. [ 10 ]…”