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Regulatory elements within the prodomain of Falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum.

Pandey KC, Barkan DT, Sali A, Rosenthal PJ - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
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.

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Model rationalizing the inhibition of cathepsin B by the falcipain-2 prodomain.(a) Model of the falcipain-2 prodomain (red) and mature cathepsin B (blue; catalytic triad residues in yellow). The prodomain binds to cathepsin B in a similar fashion as zymogens of other cysteine proteases, including procathepsin L and procathepsin B. (b) Structural overlay of mature cathepsin B (blue) and falcipain-2 (cyan). Catalytic triad residues are shown in the stick representation (yellow: cathepsin B; orange: falcipain-2). Cathepsin B amino acid numbering is used.
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pone-0005694-g006: Model rationalizing the inhibition of cathepsin B by the falcipain-2 prodomain.(a) Model of the falcipain-2 prodomain (red) and mature cathepsin B (blue; catalytic triad residues in yellow). The prodomain binds to cathepsin B in a similar fashion as zymogens of other cysteine proteases, including procathepsin L and procathepsin B. (b) Structural overlay of mature cathepsin B (blue) and falcipain-2 (cyan). Catalytic triad residues are shown in the stick representation (yellow: cathepsin B; orange: falcipain-2). Cathepsin B amino acid numbering is used.

Mentions: A separate homology model was constructed in which the falcipain-2 prodomain and cathepsin-B mature domain were modeled as a complex (Fig. 6a). The model was built based on an alignment of profalcipain-2 at 31.2% sequence identity with the crystallographic structure of procathepsin B (25, 26). The model received a DOPE Z score of −0.87, and a TSVMod native overlap prediction of 0.82. These scores indicate that the overall fold is correct; poor scores would have suggested that there were significant errors in the modeled structure of the prodomain, and in that case the model would not have resembled the structures of the templates on which it was based. The model suggests that the prodomain of falcipain-2 binds mature cathepsin B in a manner similar to that observed in papain family zymogens, inhibiting catalytic activity by blocking substrate access to the active site. (Fig. 6b). While no structure has been solved for a propeptide in complex with an inhibited mature enzyme, it is likely that these propeptides bind to the enzymes in a conformation resembling the zymogen form (14, 25–27). This hypothesis is reflected in the model, which by construction is similar to its templates, and displays favorable stereochemistry and non-bonded atom distances as evaluated by MODELLER and DOPE.


Regulatory elements within the prodomain of Falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum.

Pandey KC, Barkan DT, Sali A, Rosenthal PJ - PLoS ONE (2009)

Model rationalizing the inhibition of cathepsin B by the falcipain-2 prodomain.(a) Model of the falcipain-2 prodomain (red) and mature cathepsin B (blue; catalytic triad residues in yellow). The prodomain binds to cathepsin B in a similar fashion as zymogens of other cysteine proteases, including procathepsin L and procathepsin B. (b) Structural overlay of mature cathepsin B (blue) and falcipain-2 (cyan). Catalytic triad residues are shown in the stick representation (yellow: cathepsin B; orange: falcipain-2). Cathepsin B amino acid numbering is used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682653&req=5

pone-0005694-g006: Model rationalizing the inhibition of cathepsin B by the falcipain-2 prodomain.(a) Model of the falcipain-2 prodomain (red) and mature cathepsin B (blue; catalytic triad residues in yellow). The prodomain binds to cathepsin B in a similar fashion as zymogens of other cysteine proteases, including procathepsin L and procathepsin B. (b) Structural overlay of mature cathepsin B (blue) and falcipain-2 (cyan). Catalytic triad residues are shown in the stick representation (yellow: cathepsin B; orange: falcipain-2). Cathepsin B amino acid numbering is used.
Mentions: A separate homology model was constructed in which the falcipain-2 prodomain and cathepsin-B mature domain were modeled as a complex (Fig. 6a). The model was built based on an alignment of profalcipain-2 at 31.2% sequence identity with the crystallographic structure of procathepsin B (25, 26). The model received a DOPE Z score of −0.87, and a TSVMod native overlap prediction of 0.82. These scores indicate that the overall fold is correct; poor scores would have suggested that there were significant errors in the modeled structure of the prodomain, and in that case the model would not have resembled the structures of the templates on which it was based. The model suggests that the prodomain of falcipain-2 binds mature cathepsin B in a manner similar to that observed in papain family zymogens, inhibiting catalytic activity by blocking substrate access to the active site. (Fig. 6b). While no structure has been solved for a propeptide in complex with an inhibited mature enzyme, it is likely that these propeptides bind to the enzymes in a conformation resembling the zymogen form (14, 25–27). This hypothesis is reflected in the model, which by construction is similar to its templates, and displays favorable stereochemistry and non-bonded atom distances as evaluated by MODELLER and DOPE.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus