Extended Data Fig. 1: Impact of extracellular-intracellular (EC-IC) communication and dimerization on designed receptor signaling with inactive biased sensor.

(a) Schematic representation of the two primary structural mechanisms driving single-pass receptor activation: Pre-Formed Dimer (PFD) and Monomer-Dimer Equilibrium (MDE). In the PFD mechanism, the sensor and responder exist in a pre-formed dimer and transition between two dimeric conformations. In contrast, the MDE mechanism involves the sensor and responder switching from monomeric to dimeric conformations. Both mechanisms ultimately converge on the same activation state. (b-c) Biosensor design scenarios involving a sensor and responder, each intrinsically biased toward either the inactive or active state, and their resulting activation behavior. (b) Inactive-biased sensor and active-biased responder. Left. In the low communication mode, the biosensor’s behavior is primarily governed by the intrinsic properties of the sensor and responder domains. The responder tends to occupy the active state, leading to high basal activity even in the absence of ligand. Meanwhile, the sensor favors an inactive conformation, resulting in low apparent ligand binding affinity and a higher EC50. Due to suboptimal signal transmission between the sensor and responder, the sensitivity to ligand binding, as reflected in the hill slope, is also low. Our selected CMRs were designed according to this scenario. Right. In the high communication mode, the sensor substantially influences the responder’s behavior, shifting it toward the inactive state. Conversely, the responder pushes the sensor toward the active state, which enhances ligand binding affinity and lowers the EC50. The hill slope increases due to stronger signal transmission between the sensor and responder. Our VMRs correspond to this high communication scenario. (c) Inactive-biased sensor and inactive-biased responder. Left: In the low communication mode, basal activity remains minimal as the responder favors its inactive state. Ligand-saturating conditions fail to elicit a maximal response due to weak coupling between the sensor and responder. Right: In high communication mode, basal activity remains minimal due to the strong inactive state bias of both the sensor and responder. However, at saturating ligand conditions, maximal activity is achieved. The EC50 is higher compared to (b) and (c) because ligand binding competes with the inactive bias in both the sensor and responder. (a-c) Generated with bioRender.