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...

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel ␤-helix (L␤H), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.Uridinediphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) 2 is present in all three kingdoms of life and catalyzes the reaction of UTP ϩ glucose 1-phosphate 3 UDP-glucose ϩ PP i in the presence of Mg 2ϩ in vivo. UDP-glucose, the activated form of glucose, plays an essential role in the metabolism of carbohydrates in all organisms. UDP-glucose is the main glucosyl donor for the formation of glycogen, starch, and cellulose, as well as for the synthesis of glucose-containing glycolipids, glycoproteins, and proteoglycans (1, 2). In addition, other important nucleotide sugars such as UDP-xylose, UDP-glucuronic acid, and UDP-galactose are derived from UDP-glucose. In bacteria some of these activated sugars are used to build the bacterial polysaccharide capsule that often represents the sole determinant of virulence of these organisms. In Streptococcus pneumoniae, for example, it was known that mutants containing an inactivated UGPase gene (galU) are completely avirulent, as they are unable to form the polysaccharide capsule (3). Similarly, UGPase is involved in the biosynthesis of the lipopolysaccharide core in Escherichia coli, resulting in a reduced adhesion behavior of E. coli galU mutants (4).The protozoan parasite Leishmania is the causative agent of a widespread group of diseases collectively known as Leishmaniasis. The disease affects more than 12 million people worldwide and until now there is no specific drug available to cure the disease.3 Leishmania express various glycoconjugates on their cell surface that is dynamically modified during the parasite life cycle allowing the survival and proliferation in the sand fly vector as well as in the mammalian host (6, 7).The biosynthesis of glycoconjugates essentially depends on the availability of activated nucleotide sugars. UGPase represents a key position in...

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one β-propeller and α/β hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.