Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.
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.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.Show MeSH
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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.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.