Limits...
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.

Show MeSH

Related in: MedlinePlus

Modeled differences between falcipain-2 (a) and cathepsin L (b) prodomain binding to cathepsin B.The model predicts a helix arrangement in the falcipain-2 prodomain (purple) that prevents steric clashes with the cathepsin B occluding loop (cyan). Phe186 may mediate this arrangement; in cathepsin K and cathepsin L, Phe186 is replaced by Arg. For cathepsin L (red), there is a large steric clash between the linker joining the two cathepsin L helices and the space-filled occluding loop (cyan).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2682653&req=5

pone-0005694-g007: Modeled differences between falcipain-2 (a) and cathepsin L (b) prodomain binding to cathepsin B.The model predicts a helix arrangement in the falcipain-2 prodomain (purple) that prevents steric clashes with the cathepsin B occluding loop (cyan). Phe186 may mediate this arrangement; in cathepsin K and cathepsin L, Phe186 is replaced by Arg. For cathepsin L (red), there is a large steric clash between the linker joining the two cathepsin L helices and the space-filled occluding loop (cyan).

Mentions: Cathepsin B activity is inhibited by the prodomain of falcipain-2 (Fig. 3) but not cathepsin L (10). To examine the structural basis of this selectivity, we compared the sequences and structures of these two proteins. Several differences were of note (Fig. 7). First, while the procathepsin L α1 helix clashes with the occluding loop region of mature cathepsin B, thus preventing binding, the equivalent helix in falcipain-2 does not. Second, Phe186 in profalcipain-2 participates in polar interactions with Phe165 and Phe168; in procathepsin K and procathepsin L, Phe186 is replaced by Arg. Third, a multiple sequence alignment reveals a conserved motif (LMNNAEHIN in falcipain-2) in the plasmodial proteases falcipain-2, falcipain-3, and berghepain-2 that represents an insertion relative to the sequences of procathepsin K and procathepsin L (Fig. 1). Finally, an apparent salt bridge (interaction not shown) is formed between Glu210 in the falcipain-2 prodomain and Lys184 in mature cathepsin B; Glu210 of falcipain-2 (which has replaced Gly in the GNFD motif) is replaced by Ala in cathepsin L and cathepsin K. Taken together, differences between modeled interactions for the cathepsin B mature domain with procathepsin L or profalcipain-2 appear to describe the structural basis for the observed selective inhibition of cathepsin B activity by profalcipain-2.


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)

Modeled differences between falcipain-2 (a) and cathepsin L (b) prodomain binding to cathepsin B.The model predicts a helix arrangement in the falcipain-2 prodomain (purple) that prevents steric clashes with the cathepsin B occluding loop (cyan). Phe186 may mediate this arrangement; in cathepsin K and cathepsin L, Phe186 is replaced by Arg. For cathepsin L (red), there is a large steric clash between the linker joining the two cathepsin L helices and the space-filled occluding loop (cyan).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005694-g007: Modeled differences between falcipain-2 (a) and cathepsin L (b) prodomain binding to cathepsin B.The model predicts a helix arrangement in the falcipain-2 prodomain (purple) that prevents steric clashes with the cathepsin B occluding loop (cyan). Phe186 may mediate this arrangement; in cathepsin K and cathepsin L, Phe186 is replaced by Arg. For cathepsin L (red), there is a large steric clash between the linker joining the two cathepsin L helices and the space-filled occluding loop (cyan).
Mentions: Cathepsin B activity is inhibited by the prodomain of falcipain-2 (Fig. 3) but not cathepsin L (10). To examine the structural basis of this selectivity, we compared the sequences and structures of these two proteins. Several differences were of note (Fig. 7). First, while the procathepsin L α1 helix clashes with the occluding loop region of mature cathepsin B, thus preventing binding, the equivalent helix in falcipain-2 does not. Second, Phe186 in profalcipain-2 participates in polar interactions with Phe165 and Phe168; in procathepsin K and procathepsin L, Phe186 is replaced by Arg. Third, a multiple sequence alignment reveals a conserved motif (LMNNAEHIN in falcipain-2) in the plasmodial proteases falcipain-2, falcipain-3, and berghepain-2 that represents an insertion relative to the sequences of procathepsin K and procathepsin L (Fig. 1). Finally, an apparent salt bridge (interaction not shown) is formed between Glu210 in the falcipain-2 prodomain and Lys184 in mature cathepsin B; Glu210 of falcipain-2 (which has replaced Gly in the GNFD motif) is replaced by Ala in cathepsin L and cathepsin K. Taken together, differences between modeled interactions for the cathepsin B mature domain with procathepsin L or profalcipain-2 appear to describe the structural basis for the observed selective inhibition of cathepsin B activity by profalcipain-2.

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