Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.

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The aspartate-specific caspases play a pivotal role in the execution of programmed cell death and therefore constitute important targets for the control of apoptosis. Upon ectopic expression, baculovirus P35 inhibits apoptosis in phylogenetically diverse organisms by suppressing the proteolytic activity of the cellular caspases in a cleavage-dependent mechanism. After cleavage by caspase, the P35 fragments remain bound to the target caspase, forming an inhibitory complex that sequesters the caspase from further activity. Crystals of a complex between P35 and Sf-caspase-1, an insect effector-caspase, were grown. A 5.2 A resolution structure of this inhibitory complex was determined by molecular-replacement methods. The structure reveals few regions of interaction between the two proteins, much like that observed in the structure of the recently solved human initiator-caspase/P35 complex. In the effector-caspase/P35 complex structure presented here, the P35 molecule shifts towards a loop that is conserved in effector caspases but absent in initiator caspase. This shift could strengthen interactions between the two proteins and may explain the preference of P35 for inhibiting effector-caspases.

Caspases play an essential role in the execution of apoptosis. These cysteine proteases are highly conserved among metazoans and are translated as inactive zymogens, which are activated by proteolytic cleavages to generate the large and small subunits and remove the N-terminal prodomain. The 2.3 Å resolution crystal structure of active Sf-caspase-1, the principal effector caspase of the insect Spodoptera frugiperda, is presented here. The structure represents the first nonhuman caspase to be resolved. The structure of the cleaved and active protease was determined with the tetrapeptide inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone covalently bonded to the active site cysteine. As expected, the overall fold of Sf-caspase-1 is exceedingly similar to that of the five active caspases from humans solved to date. The overall structure and active site arrangement of Sf-caspase-1 is most comparable with that of the human effector caspases, with which it shares highest sequence homology. The most prominent structural difference with Sf-caspase-1 is the position of the N-terminal region of the large subunit. Unlike the N terminus of human caspases, the N terminus of Sfcaspase-1 originates from the active site side where it interacts with active site loop L2 and then extends to the backside of the heterodimer. This unusual structural arrangement raises the possibility that the N-terminal prodomain plays a regulatory role during effector caspase activation or enzyme activity in insects.Apoptosis is a cellular pathway that eliminates damaged, potentially dangerous, superfluous, or unwanted cells in metazoan organisms. This programmed cell death pathway is a naturally occurring physiological process that is vital to normal organismal development and tissue homeostasis. Abnormalities in the regulation of apoptosis can trigger many diseases including cancer, neurodegenerative disorders, autoimmune disorders, and ischemic injury. The apoptotic pathway is highly conserved in the Metazoa kingdom and can be triggered by both intracellular and extracellular stimuli. Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases (1-6). Because of their central role in apoptosis, these cysteine proteases are attractive targets for therapeutic intervention.Synthesized as dormant single-chain zymogens, the caspases are activated by a hierarchical series of proteolytic cleavages. The apical initiator caspases typically contain a large N-terminal prodomain that interacts with cellular factors that initiate apoptosis. These initiator caspases oligomerize through their prodomains that also associate with other proteins and promote enzyme activation in the absence of proteolytic processing (7). The activated initiator caspases subsequently cleave the proform of downstream effector caspases, which usually possess a short N-terminal prodomain. The active effector caspases proteolytically cleave cellular substrates that produce the morphological events associated with apoptosis, including cytoplasmic shr...

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