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 seek further insights into the binding determinants of the SERA4st1 substrate, we performed an unrestrained MD simulation of the PfSUB1–SERA4st1 complex in explicit solvent. Determining the RMSF of backbone atoms of the bound peptide revealed that pronounced conformational changes occur at the P2′–P5′ prime side residues, whereas the remaining part of the peptide backbone remain essentially unchanged (Fig. 5A and B). The restricted fluctuation of the P5–P1′ residues is in agreement with strong backbone-backbone hydrogen bonds being formed between P2′–P4 and the PfSUB1 binding site (Fig. 3). Over the course of the 50 ns of simulation time, very stable canonical hydrogen bonds (with an occupancy of at least 80%, indicated in red in Fig. 5C) are formed between the amino group of P2′ and the carbonyl group of Asn603 in the S2′ pocket as well as between the amino and carbonyl group of P4 with the corresponding backbone groups of Gly467. Further canonical hydrogen bonds (with an occupancy of at least 40%, indicated in orange or green in Fig. 5C) are formed between the carbonyl group of P1 and Thr605 and Ser606, respectively, between the amino group of P1 and Ser490, and between the amino group of P2 and Lys465, respectively. In addition, stabilizing hydrogen bond interactions are formed between the side-chain carboxylate group of P1 and the backbone amide group of Asn520 in the S1 pocket, between the side-chain keto group of P1′ and the side chain ε-amino group of Lys465 in the S2 pocket, and between the side-chain hydroxyl group of P3′ and the side-chain guanidinium group of Arg600.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.