Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition.The development and homeostasis of multicellular organisms depend on a delicate balance of cell proliferation and programmed cell death or apoptosis. Failure to control either of these processes can lead to serious diseases that threaten the existence of the organism (1, 2). For example, the down-regulation of apoptosis is often associated with cancer, autoimmune disorders, and persistent viral infections. The up-regulation of apoptosis is observed in many forms of degenerative disorders such as Alzheimer's disease, ischemic injury from stroke, and post-menopausal osteoporosis.The central effectors of apoptotic cell death are caspases, a group of cysteine proteases specific for aspartate residues (3). Caspases are highly regulated at several different levels. First, they are synthesized as inactive single-chain zymogens. Second, caspase activation is achieved through controlled proteolytic cascades, with upstream caspases (Group III, such as caspase-8 and caspase-9) activated by signal-mediated oligomerization and autoprocessing and downstream caspases (Group II, such as caspase-3 and casapse-7) activated by upstream caspases. While caspases have a dominant requirement for Asp at the P1 position, neighboring sequences at P5-P1Ј (in particular P4-P2) influence the substrate specificity of each group of caspases (4). Active caspases are in addition subject to inhibition by specific viral and cellular caspase inhibitors (5, 6). Most notably, the p35 protein from baculoviruses is an effective and the only wide-spectrum caspase inhibitor. It blocks apoptosis induced by numerous stimuli and in diverse organism...