Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum

Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu155–Asp243) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain

A unique Oct4 interface is crucial for reprogramming to pluripotency

Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein's surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein-protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process

Proteases universally recognize beta strands in their active sites

One way to combat infectious diseases is to selectively inhibit foreign proteases within host cells, thus retarding replication rates of infectious organisms and assisting normal immunological defense mechanisms involved in their eradication. This review gives a summary of over 1500 three dimensional crystal (X-ray) and solution (NMR) structures from the pdb of substrates, products and inhibitors bound in the active sites of aspartic, serine, metallo, cysteine, and threonine endopeptidases. These active sites of all five protease classes recognize peptidic and non-peptidic ligands in an extended beta strand conformation, with few exceptions. Comparisons of protease-bound ligand conformations are illustrated by structural superpositions for a subset of structures, including 21 aspartic, 44 serine, 20 metallo, 23 cysteine, and 2 threonine proteases, among the protease-ligand structures analyzed. The extended substrate-binding mode is also illustrated for 3 aspartic proteases, 1 serine protease, 1 cysteine protease and 1 metalloprotease