Abstract
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.
Highlights
Malaria is one of the most important infectious diseases in the world, infecting hundreds of millions of people and causing over one million deaths each year [1]
We recently showed that this amino-terminal extension is required for folding to active enzyme and that it can mediate this process either when covalently bound to the catalytic domain or when included in refolding buffer as part of a separate prodomain-folding domain construct [18]
Mediators of Folding, Activity, and Inhibition of Falcipain-2 with this domain? Third, is the amino-terminal extension required only for folding, or does it play an essential role in mediating enzyme activity? Fourth, once folding information is imparted on the catalytic domain, are intact disulfide bonds sufficient to direct refolding? Fifth, is the mediation of folding domain activity by the prodomain a consequence of interaction of the prodomain with the active site? We report a series of mutagenesis studies that have answered these questions and shown that enzyme activity, folding, and inhibition are each mediated by different domains of falcipain-2 and that the interaction of these domains appears to be optimized for tight control of proteolytic activity
Summary
Amplification and Cloning of Expression Constructs—Constructs were amplified using vent DNA polymerase (New England Biolabs) and the primers listed below. The DNA fragments were digested with BamHI and HindIII, ligated into appropriately digested plasmids (pQE30; Qiagen), and used to transform M15 (pREP4) Escherichia coli (Qiagen). For falcipain-3, the DNA fragments were digested with SalI and BamHI, ligated into digested pRESETA (Qiagen) plasmids, and used to transform AD494 (DE3) pLysS E. coli (Novagen). For knowlepain-2, the fragments were digested with BamHI and PstI, ligated into digested pQE-30, and used to transform M15 (pREP4) E. coli. The DNA fragments were digested with BamHI and HindIII, ligated into digested pQE-30, and used to transform M15 (pREP4) E. coli, except for the chimeric construct of berghepain-2, in which the pRESET-A vector and BL21(DE3) E. coli were used. The rate of hydrolysis of Z-Leu-ArgAMC in the presence of constant enzyme concentration and varying substrate concentrations was determined at room temperature in 100 mM sodium acetate, pH 5.5, 8 mM DTT. The nitrocellulose membranes were blocked with 2% bovine serum albumin overnight at 4 °C, washed with phosphate-buffered saline with 0.05% Tween 20 (PBST), incubated with rat antiserum raised against the falcipain-2 prodomain (1:1500 dilution) for 60 min at room temperature, washed with PBST, incubated with alkaline-phosphatase-conjugated goat antirat IgG (Sigma) at 1:2000 dilution for 60 min at room temperature, washed with PBST, and developed with 5-bromo-4-chloro-3-indolyl
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