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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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Homology modelling and recombinant expression reveals similarities and differences between Plasmodium subtilisin-like protease 1 (SUB1) orthologues. (A) Molecular surface representation of homology models of the Plasmodium vivax (Pv)SUB1, Plasmodium knowlesi (Pk)SUB1 and Plasmodium berghei (Pb)SUB1 catalytic domains. Coloured residues (blue for PvSUB1, green for PkSUB1, red for PbSUB1) indicate positions that are different from structurally equivalent residues in PfSUB1, whereas identical residues are shown in grey. The catalytic Ser is shown in yellow. White dashed frames delimitate the active site grooves, and the zoomed views below show the modelled binding mode of SERA4st1 (KITAQ ↓ DDEES). PvSUB1 and PkSUB1 are almost identical to PfSUB1 in the active site region, with the only two substitutions being Ala481 and Gln407 in PvSUB1, and Ala490 and Gln416 in PkSUB1. In contrast, PbSUB1 diverges significantly, with six substitutions (His340, Lys378, Ile386, Met513, Glu514 and Ser516), indicating likely differences in the specificity of this enzyme at the P4 and P′ positions. (B) Recombinant expression of SUB1 orthologues. Proteins were expressed using baculovirus-infected insect cells and purified as described in Section 2.1. Positions of migration of the protease domain and the prodomain species resulting from cleavage during maturation are indicated. The left-hand track on each gel contains molecular mass standards of the indicated sizes. Predicted molecular masses of the protease domains resulting from cleavage at the expected autocatalytic processing site (Supplementary Fig. S1) are: PvSUB1, 48,082 Da; PkSUB1, 49,072 Da; and PbSUB1, 44,868 Da.
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f0035: Homology modelling and recombinant expression reveals similarities and differences between Plasmodium subtilisin-like protease 1 (SUB1) orthologues. (A) Molecular surface representation of homology models of the Plasmodium vivax (Pv)SUB1, Plasmodium knowlesi (Pk)SUB1 and Plasmodium berghei (Pb)SUB1 catalytic domains. Coloured residues (blue for PvSUB1, green for PkSUB1, red for PbSUB1) indicate positions that are different from structurally equivalent residues in PfSUB1, whereas identical residues are shown in grey. The catalytic Ser is shown in yellow. White dashed frames delimitate the active site grooves, and the zoomed views below show the modelled binding mode of SERA4st1 (KITAQ ↓ DDEES). PvSUB1 and PkSUB1 are almost identical to PfSUB1 in the active site region, with the only two substitutions being Ala481 and Gln407 in PvSUB1, and Ala490 and Gln416 in PkSUB1. In contrast, PbSUB1 diverges significantly, with six substitutions (His340, Lys378, Ile386, Met513, Glu514 and Ser516), indicating likely differences in the specificity of this enzyme at the P4 and P′ positions. (B) Recombinant expression of SUB1 orthologues. Proteins were expressed using baculovirus-infected insect cells and purified as described in Section 2.1. Positions of migration of the protease domain and the prodomain species resulting from cleavage during maturation are indicated. The left-hand track on each gel contains molecular mass standards of the indicated sizes. Predicted molecular masses of the protease domains resulting from cleavage at the expected autocatalytic processing site (Supplementary Fig. S1) are: PvSUB1, 48,082 Da; PkSUB1, 49,072 Da; and PbSUB1, 44,868 Da.

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.


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)

Homology modelling and recombinant expression reveals similarities and differences between Plasmodium subtilisin-like protease 1 (SUB1) orthologues. (A) Molecular surface representation of homology models of the Plasmodium vivax (Pv)SUB1, Plasmodium knowlesi (Pk)SUB1 and Plasmodium berghei (Pb)SUB1 catalytic domains. Coloured residues (blue for PvSUB1, green for PkSUB1, red for PbSUB1) indicate positions that are different from structurally equivalent residues in PfSUB1, whereas identical residues are shown in grey. The catalytic Ser is shown in yellow. White dashed frames delimitate the active site grooves, and the zoomed views below show the modelled binding mode of SERA4st1 (KITAQ ↓ DDEES). PvSUB1 and PkSUB1 are almost identical to PfSUB1 in the active site region, with the only two substitutions being Ala481 and Gln407 in PvSUB1, and Ala490 and Gln416 in PkSUB1. In contrast, PbSUB1 diverges significantly, with six substitutions (His340, Lys378, Ile386, Met513, Glu514 and Ser516), indicating likely differences in the specificity of this enzyme at the P4 and P′ positions. (B) Recombinant expression of SUB1 orthologues. Proteins were expressed using baculovirus-infected insect cells and purified as described in Section 2.1. Positions of migration of the protease domain and the prodomain species resulting from cleavage during maturation are indicated. The left-hand track on each gel contains molecular mass standards of the indicated sizes. Predicted molecular masses of the protease domains resulting from cleavage at the expected autocatalytic processing site (Supplementary Fig. S1) are: PvSUB1, 48,082 Da; PkSUB1, 49,072 Da; and PbSUB1, 44,868 Da.
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f0035: Homology modelling and recombinant expression reveals similarities and differences between Plasmodium subtilisin-like protease 1 (SUB1) orthologues. (A) Molecular surface representation of homology models of the Plasmodium vivax (Pv)SUB1, Plasmodium knowlesi (Pk)SUB1 and Plasmodium berghei (Pb)SUB1 catalytic domains. Coloured residues (blue for PvSUB1, green for PkSUB1, red for PbSUB1) indicate positions that are different from structurally equivalent residues in PfSUB1, whereas identical residues are shown in grey. The catalytic Ser is shown in yellow. White dashed frames delimitate the active site grooves, and the zoomed views below show the modelled binding mode of SERA4st1 (KITAQ ↓ DDEES). PvSUB1 and PkSUB1 are almost identical to PfSUB1 in the active site region, with the only two substitutions being Ala481 and Gln407 in PvSUB1, and Ala490 and Gln416 in PkSUB1. In contrast, PbSUB1 diverges significantly, with six substitutions (His340, Lys378, Ile386, Met513, Glu514 and Ser516), indicating likely differences in the specificity of this enzyme at the P4 and P′ positions. (B) Recombinant expression of SUB1 orthologues. Proteins were expressed using baculovirus-infected insect cells and purified as described in Section 2.1. Positions of migration of the protease domain and the prodomain species resulting from cleavage during maturation are indicated. The left-hand track on each gel contains molecular mass standards of the indicated sizes. Predicted molecular masses of the protease domains resulting from cleavage at the expected autocatalytic processing site (Supplementary Fig. S1) are: PvSUB1, 48,082 Da; PkSUB1, 49,072 Da; and PbSUB1, 44,868 Da.
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.

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