Predicted interactions between the prodomain and the mature domain of FP2 and FP3.

<p><b>A.</b> Close up of predicted interactions between the mature enzyme and the ERFNIN and GNFD motifs of the prodomain (Arg ¹⁸⁵ - Glu ²²¹, and Phe ²¹⁴-Trp⁴⁴⁹/Trp ⁴⁵³, Glu ²¹⁰ - Lys ⁴⁰³). Blue dashed lines indicate presumed stabilizing interactions between residues in FP2. <b>B.</b> Blue dashed lines indicate presumed stabilizing interactions (Arg ²⁰²-Glu²³⁸ and Phe ²³¹-Trp⁴⁵⁷/Trp461) between the residues in FP3.</p

Role of salt bridge and hydrophobic interactions in FP2.

<p><b>A.</b> The mutated enzyme (Glu ²²¹) was expressed in <i>E.Coli</i>, purified and refolded, and finally processed and compare with wild FP2, analyzed by SDS-PAGE and Western blot. <b>B.</b> Two other mutants (Glu ²¹⁰ and Phe ²¹⁴) were also expressed in <i>E.coli</i>, purified and refolded. The processing of those mutants was further analyzed by SDS-PAGE and Western blot analysis. The uninduced <i>E.Coli</i> lysates was used as a negative control. <b>C.</b> The wild FP2 and mutated enzymes (Glu ²²¹, Glu ²¹⁰, Asp ¹⁵¹, Phe ²¹⁴) were processed and subjected to gelatin substrate native PAGE, and assessed their functional activity.</p

Trans processing of the Pro-FP3 mutant.

<p>Mutants of FP3 were incubated with active FP2 under optimal conditions for trans processing. For digestion, the pro-FP3 mutants (10 µg) were suspended in acetate buffer and 5 µg of active FP2 was mix with each reaction and incubated at room temperature. The wild Pro-FP3, which was auto processed used as control. Digestion products were analysed by SDS-PAGE and western blot analysis using specific antibodies against FP3.</p

Auto-activation of Pro-FP2.

<p>The activation of Pro-FP2 was represented by complex of prodomain (cyan) and mature domain (Purple). Structural features of inactive and active FP2 were shown, where 160 N-terminal residues of the prodomain was not included in the model.</p

Alignment of C-terminus amino acid residues of the prodomains of FP2, FP3, and related papain family cysteine proteases.

<p><b>A.</b> The sequences of FP2, FP3, berghepain-2 (BP2), and human cysteine proteases, cathepsin K (Cath K), cathepsin L (Cath L), cathepsin B (Cath B), and papain were aligned using Gene doc and Clustal W. The mutated amino acids are shown in red, and highly conserved amino acids are highlighted in black. <b>B.</b> The Mutant (Asp¹⁵¹) was purified and refolded and processed under optimized acidic condition as described in material methods. The protein was resolved by SDS-PAGE, and analyzed by Western blot analysis. The positions of molecular weight markers (kDa) are indicated.</p

Sequences of the primers used in this study, mutated sites are in bold type.

<p>Sequences of the primers used in this study, mutated sites are in bold type.</p

Model showing mechanism of activation of falcipain.

<p>The prodomain (purple) and the mature domain (cyan) are stabilized by salt and hydrophobic interactions (shown by yellow dashes). The wild enzyme (upper panel) processed normally in acidic condition where the prodomain is released and the mature domain is catalytically active. On the other hand, mutants (mutations indicated by red labeled) with disrupted salt and hydrophobic interactions failed to process into active enzyme.</p

Requirements for ionic and hydrophobic interactions for processing of Pro-FP3.

<p><b>A.</b> The wild FP3 and mutated enzyme, Glu ²³⁸ and Phe ²³¹ were expressed in <i>E.Coli</i>, purified and refolded, and finally processed and compare with wild FP3 . The enzymes were further analyzed by SDS-PAGE and Western blot. <b>B.</b> The wild FP3 and mutated enzymes (Phe ²³¹, Glu ²³⁸) were processed and subjected to gelatin substrate native PAGE, and assess their functional activity.</p

Expression, purification, refolding and processing of Pro-FP2.

<p><b>A.</b> Purified and refolded Pro-FP2 was activated/processed in acidic condition. The processing of enzyme was followed by 3 hrs and further evaluated by SDS-PAGE and Western blot analysis. The approximate size of Pro-FP2 and FP2 were mentioned in the figure. <b>B.</b> Purified and refolded Pro-FP2 was processed in the presence of different inhibitors. Leupeptin, E-64 and, prodomain of FP2, were used as a cysteine proteases inhibitors. Whereas, PMSF (phenylmethylsulfonyl fluoride), pepstatin and EDTA were used as serine, aspartic and mettalo proteases, respectively. Finally, proteins were evaluated by SDS-PAGE and western blot analysis.</p

Alignment of falstatin with other ICPs.

<p>A multiple sequence alignment was performed with falstatin and ICPs from <i>Cryptosporidium parvum</i> (cryptostatin), <i>Trypanosoma cruzi</i> (chagasin), and <i>Plasmodium berghei</i> (PbICP). This alignment predicted four major loop regions; L0, BC (L2), DE (L4) and FG (L6) in falstatin. The peptides used in this study are underlines, and residues mutated in the described studies are in bold type.</p