No abstract

Caspases form a family of proteinases required for the initiation and execution phases of apoptosis. Distinct proapoptotic stimuli lead to activation of the initiator caspases-8 and -9, which in turn activate the common executioner caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures of several active caspases have been reported, no three-dimensional structure of an uncleaved caspase zymogen is available so far. We have determined the 2.9-Å crystal structure of recombinant human C285A procaspase-7 and have elucidated the activation mechanism of caspases. The overall fold of the homodimeric procaspase-7 resembles that of the active tetrameric caspase-7. Each monomer is organized in two structured subdomains connected by partially flexible linkers, which asymmetrically occupy and block the central cavity, a typical feature of active caspases. This blockage is incompatible with a functional substrate binding site͞active site. After proteolytic cleavage within the flexible linkers, the newly formed chain termini leave the cavity and fold outward to form stable structures. These conformational changes are associated with the formation of an intact active-site cleft. Therefore, this mechanism represents a formerly unknown type of proteinase zymogen activation. P rogrammed cell death (apoptosis) is associated with the hierarchical activation of a number of cysteine proteinases with cleavage preference after Asp residues, comprising the caspase family C14 of clan CD (1). Based on their position within this proteolytic cascade, caspases are subdivided into initiator caspases (including caspases-8 and -9) and executioner caspases (including caspases-3, -6, and -7), as well as a third group of caspases (caspases-1, -4, and -5) involved in cytokine activation. In apoptosis, the upstream caspases triggered by cofactormediated trans-activation activate the downstream executioner caspases by limited proteolysis (2-4). These executioners, in turn, cleave distinct intracellular proteins involved in promoting the apoptotic phenotype.Because of the potentially hazardous action of activated caspases in the host cell, effective mechanisms must keep them in a latent form before activation. Both the initiator and the executioner caspases are initially synthesized as single-chain molecules, most of which require proteolytic cleavage in their C-terminal half (physiologically at a distinct Asp-Xxx scissile bond) to become proteolytically active (5). Procaspase-7, in particular, is expressed as a 303-aa residue polypeptide chain. Upon activation in vivo, a short N-terminal peptide is removed, and, more importantly from the perspective of generating catalytic activity, an Ile-Gln-Ala-Asp-2-Ser-Gly site is cleaved, giving rise to a 175-residue large chain and a 105-residue small chain, comprising the active caspase-7. The only known exception to this mode of activation is caspase-9, whose zymogen does not require proteolytic processing for activity (6, 7).The first crystal structures of active caspase-1 (8, 9), and later th...

Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the ␥ fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.