Ras, a small GTPase, functions as a molecular switch in diverse signaling pathways, controlling processes such as cell growth and differentiation. Its activity is determined by the nucleotide state—Ras-GTP represents the active “ON” state, while hydrolysis to GDP switches it “OFF”. This cycle is modulated by GAPs and GEFs. A key step in downstream signaling is the interaction between Ras-GTP and effector proteins through the Ras-binding domain (RBD), which binds Ras-GTP with nanomolar affinity at a conserved interface. We hypothesized that artificial regulation of this interaction could enable external control of Ras signaling. To achieve this, we engineered a Ca 2+ -responsive system by fusing calmodulin (CaM) and the M13 peptide to the termini of RBD. CaM undergoes conformational rearrangements upon Ca 2+ binding and specifically recognizes M13 with high affinity. Structural modeling using AlphaFold3 predicted that, under Ca 2+ -bound conditions, CaM-M13 interaction sterically occludes the Ras-binding site of RBD. Consistently, biochemical assays confirmed that Ras-RBD association was significantly inhibited in the presence of Ca 2+ . These findings demonstrate a novel ion-controlled mechanism for regulating Ras activity. Our CaM/M13-fused RBD system provides a proof-of-concept for designing ionochromic switches to modulate small GTPase signaling pathways, suggesting potential applications in synthetic biology and signal regulation.
Zhang et al. (Sun,) studied this question.