Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety.

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM-dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.

Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain.

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to ß-strand (-Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.