Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2.
The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.
Pandey
Kailash C
KC
Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143, USA.
Sijwali
Puran S
PS
Singh
Ajay
A
Na
Byoung-Kuk
BK
Rosenthal
Philip J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D007976:Leupeptins / Q000737:chemistry; D008969:Molecular Sequence Data; D010961:Plasmodium / Q000201:enzymology; D010963:Plasmodium falciparum / Q000201:enzymology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
D004220:Disulfides; D007976:Leupeptins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D003546:Cysteine Endopeptidases; C416224:falcipain 2; C032854:leupeptin