Electrostatic Clamp and Loop Dynamics Dictate Caspase-8 Cleavage of the Apoptotic Protein Bid

The canonical LQTD motif (residues 56-59) has long been regarded as the sole "address tag" that directs Caspase-8 to cleave the pro-apoptotic protein Bid at D59. Here we overturn this view, showing that Bid's previously uncharted 42-residue "disordered" loop furnishes a second layer of control that can either accelerate or block cleavage. Alanine scanning, MD simulations, DEER spectroscopy, and AlphaFold reveal two physical modules in this loop: an electrostatic clamp formed by E53-D54-E55 that secures the substrate upstream of LQTD, and an entropic flexibility switch at S61-Q62 that modulates catalysis. Phosphorylation at S61 locks the switch and halts proteolysis, whereas a double-alanine substitution (S61A-Q62A) loosens intraloop hydrogen bonding and boosts cleavage by ∼50%. Loop charge, dynamics, and post-translational modification thus cooperate to set Caspase-8 specificity, establishing dynamic loops as tunable physical targets for chemical control of apoptotic signaling.