Abstract
Calmodulin (CaM) is a Ca2+-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic ‘anchor’ residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca2+-binding motifs that form a Ca2+-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca2+-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca2+-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.
Highlights
Calmodulin (CaM) is an exceptionally highly conserved 16.7 kDa acidic protein that senses Ca2+ and interacts with and regulates a wide variety of target proteins including enzymes, kinases and phosphatases, ion channels and pumps, and transcription factors [1].The 148-amino acid sequence of CaM contains four Ca2+-binding EF-hands, each of which is a 29-a.a. helix-loop-helix motif
Ca2+ binding induces a conformational change in each lobe, which leads to the exposure of methionine-rich hydrophobic pockets that engage hydrophobic side chains of target proteins
Changes in FRET efficiency (Figure 3F) and fluorescence-lifetime imaging (FLIM) were observed upon Ca2+-induced translocation of ‘wild-type’ CaMeRAS from the membrane to the cytoplasm [55]. This was observed in different cell types using different ligands to elicit the Ca2+ signal, which was simultaneously monitored with the fluorescent Ca2+-indicator Calbryte 630, confirming that CaMeRAS is a robust biosensor and supporting the model that Ca2+-CaM binding to the KRAS4b farnesyl group extracts it from the plasma membrane [55]
Summary
Calmodulin (CaM) is an exceptionally highly conserved 16.7 kDa acidic protein that senses Ca2+ and interacts with and regulates a wide variety of target proteins including enzymes, kinases and phosphatases, ion channels and pumps, and transcription factors [1]. Sci. 2020, 21, 2751 contains a helix and hairpin that reduces the spatial distance between the anchors, which are more widely spaced in sequence [14] These are referred to canonical CaM target motifs, structures have been solved of CaM binding to many other non-canonical sequences. Ca2+-CaM can adapt to bind a short “1-3” motif in myristoylated alanine-rich C kinase substrate (MARCKS) (Figure 1) This region, which is a non-myristoylated internal peptide, is unstructured with both anchors displayed on a single helical turn. If CaM interacts with the myristoylated Src N-terminus in a manner similar to CAP23/NAP22, the structure of the latter indicates that the sequestration of the lipid and potentially the polybasic residues is likely the underlying mechanism
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