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: To investigate potentially informative structural similarities and differences between the active site architectures of SUB1 orthologues from other clinically and experimentally important malarial species, we built homology models of the catalytic domains of PkSUB1, PvSUB1 and PbSUB1, for comparison with the PfSUB1 model. This revealed (Fig. 7A) that the active sites of PvSUB1 and PkSUB1 are identical to each other and very similar to that of PfSUB1, the only important difference being the replacement of Ser537 at the base of the PfSUB1 S1 pocket with Ala in PvSUB1 and PkSUB1 (see also Supplementary Table S1). This would be predicted to result in a slightly less polar S1 pocket in these enzymes. In addition, Glu463 below the PfSUB1 S2 pocket is replaced by a Gln residue in PvSUB1 and PkSUB1, but this lies some distance from the substrate interactions so it is anticipated to have little if any effect on the conformation of the S2 pocket. In contrast, an examination of the modelled PbSUB1 active site indicated more structural divergence, with a total of six substitutions relative to PfSUB1. Notable amongst these is Ile386 at the base of the S4 pocket, which replaces a Met residue in PfSUB1, PvSUB1 and PkSUB1, and results in a slight increase in the size and hydrophobicity of the PbSUB1 S4 pocket. The PbSUB1 S′ sub-site composition is even more divergent, with Ser516 replacing Asn603 in PfSUB1, and Met513 and Glu514 replacing, respectively, Arg600 and Lys601 in PfSUB1. Combined, these substitutions are predicted to provide PbSUB1 with a less basic S′ surface than the other three orthologues, evident from a comparison of the electrostatic surface views (Supplementary Fig. S5). In summary, modelling of the SUB1 orthologues suggests that PvSUB1 and PkSUB1 likely share very similar substrate specificity with PfSUB1, whilst some differences in the specificity of PbSUB1 at the P4 and P′ positions are likely.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.