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 (Leu(155)-Asp(243)) 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.
Pandey
Kailash C
KC
Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.
Barkan
David T
DT
Sali
Andrej
A
Rosenthal
Philip J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000737:chemistry; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000737:chemistry; D002942:Circular Dichroism; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004795:Enzyme Stability; D016778:Malaria, Falciparum / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010271:Parasites / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D011498:Protein Precursors / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D010446:Peptide Fragments; D011498:Protein Precursors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C416224:falcipain 2; D002401:Cathepsin B; D056668:Cathepsin L