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Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

Withers-Martinez C, Suarez C, Fulle S, Kher S, Penzo M, Ebejer JP, Koussis K, Hackett F, Jirgensons A, Finn P, Blackman MJ - Int. J. Parasitol. (2012)

Bottom Line: Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif.Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates.Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.

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Related in: MedlinePlus

Structural model of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) catalytic domain. Cartoon representation of the PfSUB1 homology model, which extends from P. falciparum 3D7 PfSUB1 residues 339–662, superimposed onto X-ray crystal structures of seven bacterial subtilisins. These are: subtilisin DY from Bacillus licheniformis (subtilisin Carlsberg) inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone (PDB 1BH6, pink); Bacillus sp. Ak.1 subtilisin (1DBI, light blue); Bacillus lentus subtilisin (1GCI, yellow); Thermoactinomyces vulgaris subtilisin (thermitase) (1THM, orange); Bacillus licheniformis subtilisin inhibited by turkey ovomucoid third domain (OMTKY3) (1R0R, green); Bacillus mesentericus subtilisin inhibited by eglin-C (1MEE, red); and subtilisin BPN′ from Bacillus amyloliquefaciens (subtilisin Novo) inhibited by chymotrypsin inhibitor 2) (1TO2, purple). The PfSUB1 homology model is depicted in its see-through molecular envelope (grey). The active site catalytic Ser side chain is shown as a yellow stick. The six surface ‘loops’ of PfSUB1, which are absent from the bacterial homologues, are clearly visible. The vertical purple bar on the left indicates a length of 30 Å.
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f0010: Structural model of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) catalytic domain. Cartoon representation of the PfSUB1 homology model, which extends from P. falciparum 3D7 PfSUB1 residues 339–662, superimposed onto X-ray crystal structures of seven bacterial subtilisins. These are: subtilisin DY from Bacillus licheniformis (subtilisin Carlsberg) inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone (PDB 1BH6, pink); Bacillus sp. Ak.1 subtilisin (1DBI, light blue); Bacillus lentus subtilisin (1GCI, yellow); Thermoactinomyces vulgaris subtilisin (thermitase) (1THM, orange); Bacillus licheniformis subtilisin inhibited by turkey ovomucoid third domain (OMTKY3) (1R0R, green); Bacillus mesentericus subtilisin inhibited by eglin-C (1MEE, red); and subtilisin BPN′ from Bacillus amyloliquefaciens (subtilisin Novo) inhibited by chymotrypsin inhibitor 2) (1TO2, purple). The PfSUB1 homology model is depicted in its see-through molecular envelope (grey). The active site catalytic Ser side chain is shown as a yellow stick. The six surface ‘loops’ of PfSUB1, which are absent from the bacterial homologues, are clearly visible. The vertical purple bar on the left indicates a length of 30 Å.

Mentions: Fig. 2 shows the PfSUB1 homology model superimposed onto the catalytic domain structures of the seven bacterial subtilisins used as templates for our homology modelling. The high degree of structural conservation within the core of the protein surrounding the catalytic Ser is immediately apparent. Variations are seen only on peripheral loops or strands, where six large insertions are clearly noticeable in PfSUB1 outside of a 15 Å radius sphere centred on the catalytic Ser. Despite their absence from homologous bacterial subtilisins, we have shown previously that individual deletion of five of these six PfSUB1 loops severely affects rPfSUB1 maturation and/or stability of the recombinant protein in our insect cell expression system, probably due to an impact on folding (Jean et al., 2005). Of importance for the present study, however, that analysis provided no experimental evidence that any of the loops contribute to PfSUB1 substrate specificity, and indeed examination of our model is in agreement with that, suggesting that all of the loops are disposed too far from the active site to impact on substrate binding.


Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

Withers-Martinez C, Suarez C, Fulle S, Kher S, Penzo M, Ebejer JP, Koussis K, Hackett F, Jirgensons A, Finn P, Blackman MJ - Int. J. Parasitol. (2012)

Structural model of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) catalytic domain. Cartoon representation of the PfSUB1 homology model, which extends from P. falciparum 3D7 PfSUB1 residues 339–662, superimposed onto X-ray crystal structures of seven bacterial subtilisins. These are: subtilisin DY from Bacillus licheniformis (subtilisin Carlsberg) inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone (PDB 1BH6, pink); Bacillus sp. Ak.1 subtilisin (1DBI, light blue); Bacillus lentus subtilisin (1GCI, yellow); Thermoactinomyces vulgaris subtilisin (thermitase) (1THM, orange); Bacillus licheniformis subtilisin inhibited by turkey ovomucoid third domain (OMTKY3) (1R0R, green); Bacillus mesentericus subtilisin inhibited by eglin-C (1MEE, red); and subtilisin BPN′ from Bacillus amyloliquefaciens (subtilisin Novo) inhibited by chymotrypsin inhibitor 2) (1TO2, purple). The PfSUB1 homology model is depicted in its see-through molecular envelope (grey). The active site catalytic Ser side chain is shown as a yellow stick. The six surface ‘loops’ of PfSUB1, which are absent from the bacterial homologues, are clearly visible. The vertical purple bar on the left indicates a length of 30 Å.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378952&req=5

f0010: Structural model of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) catalytic domain. Cartoon representation of the PfSUB1 homology model, which extends from P. falciparum 3D7 PfSUB1 residues 339–662, superimposed onto X-ray crystal structures of seven bacterial subtilisins. These are: subtilisin DY from Bacillus licheniformis (subtilisin Carlsberg) inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone (PDB 1BH6, pink); Bacillus sp. Ak.1 subtilisin (1DBI, light blue); Bacillus lentus subtilisin (1GCI, yellow); Thermoactinomyces vulgaris subtilisin (thermitase) (1THM, orange); Bacillus licheniformis subtilisin inhibited by turkey ovomucoid third domain (OMTKY3) (1R0R, green); Bacillus mesentericus subtilisin inhibited by eglin-C (1MEE, red); and subtilisin BPN′ from Bacillus amyloliquefaciens (subtilisin Novo) inhibited by chymotrypsin inhibitor 2) (1TO2, purple). The PfSUB1 homology model is depicted in its see-through molecular envelope (grey). The active site catalytic Ser side chain is shown as a yellow stick. The six surface ‘loops’ of PfSUB1, which are absent from the bacterial homologues, are clearly visible. The vertical purple bar on the left indicates a length of 30 Å.
Mentions: Fig. 2 shows the PfSUB1 homology model superimposed onto the catalytic domain structures of the seven bacterial subtilisins used as templates for our homology modelling. The high degree of structural conservation within the core of the protein surrounding the catalytic Ser is immediately apparent. Variations are seen only on peripheral loops or strands, where six large insertions are clearly noticeable in PfSUB1 outside of a 15 Å radius sphere centred on the catalytic Ser. Despite their absence from homologous bacterial subtilisins, we have shown previously that individual deletion of five of these six PfSUB1 loops severely affects rPfSUB1 maturation and/or stability of the recombinant protein in our insect cell expression system, probably due to an impact on folding (Jean et al., 2005). Of importance for the present study, however, that analysis provided no experimental evidence that any of the loops contribute to PfSUB1 substrate specificity, and indeed examination of our model is in agreement with that, suggesting that all of the loops are disposed too far from the active site to impact on substrate binding.

Bottom Line: Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif.Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates.Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.

Show MeSH
Related in: MedlinePlus