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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

Molecular Dynamics simulation of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) structure in complex with SERA4st1. (A) Binding mode of SERA4st1 extracted after 40 ns from the MD simulation. Hydrogen bond interactions between the peptide (yellow carbon coloured stick) and PfSUB1 are indicated by dashed lines. (B) Root-mean square fluctuations (RMSF) about the mean position of the peptide backbone atoms. For RMSF calculation, overall translational and rotational motions were removed with respect to the Cα atoms of the PfSUB1 structure. (C) Scheme of hydrogen bonds formed during the MD simulation between PfSUB1 and peptide backbone and side-chain atoms. The peptide orientation is shown from N- to C-terminus left-to-right, and thus is opposite to the orientation shown in (A). Very strong hydrogen bonds (occupancy of 80–100%) are boxed in red, strong hydrogen bonds (occupancy of 60–80%) are in orange, and weaker hydrogen bonds (occupancy of 40–60%) are shown in green. Occupancy values were obtained over the course of the entire 50 ns of simulation time. Repeating the analysis for the first and second half of the trajectory resulted in the same hydrogen bond pattern.
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f0025: Molecular Dynamics simulation of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) structure in complex with SERA4st1. (A) Binding mode of SERA4st1 extracted after 40 ns from the MD simulation. Hydrogen bond interactions between the peptide (yellow carbon coloured stick) and PfSUB1 are indicated by dashed lines. (B) Root-mean square fluctuations (RMSF) about the mean position of the peptide backbone atoms. For RMSF calculation, overall translational and rotational motions were removed with respect to the Cα atoms of the PfSUB1 structure. (C) Scheme of hydrogen bonds formed during the MD simulation between PfSUB1 and peptide backbone and side-chain atoms. The peptide orientation is shown from N- to C-terminus left-to-right, and thus is opposite to the orientation shown in (A). Very strong hydrogen bonds (occupancy of 80–100%) are boxed in red, strong hydrogen bonds (occupancy of 60–80%) are in orange, and weaker hydrogen bonds (occupancy of 40–60%) are shown in green. Occupancy values were obtained over the course of the entire 50 ns of simulation time. Repeating the analysis for the first and second half of the trajectory resulted in the same hydrogen bond pattern.

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.


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)

Molecular Dynamics simulation of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) structure in complex with SERA4st1. (A) Binding mode of SERA4st1 extracted after 40 ns from the MD simulation. Hydrogen bond interactions between the peptide (yellow carbon coloured stick) and PfSUB1 are indicated by dashed lines. (B) Root-mean square fluctuations (RMSF) about the mean position of the peptide backbone atoms. For RMSF calculation, overall translational and rotational motions were removed with respect to the Cα atoms of the PfSUB1 structure. (C) Scheme of hydrogen bonds formed during the MD simulation between PfSUB1 and peptide backbone and side-chain atoms. The peptide orientation is shown from N- to C-terminus left-to-right, and thus is opposite to the orientation shown in (A). Very strong hydrogen bonds (occupancy of 80–100%) are boxed in red, strong hydrogen bonds (occupancy of 60–80%) are in orange, and weaker hydrogen bonds (occupancy of 40–60%) are shown in green. Occupancy values were obtained over the course of the entire 50 ns of simulation time. Repeating the analysis for the first and second half of the trajectory resulted in the same hydrogen bond pattern.
© Copyright Policy
Related In: Results  -  Collection

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

f0025: Molecular Dynamics simulation of the Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) structure in complex with SERA4st1. (A) Binding mode of SERA4st1 extracted after 40 ns from the MD simulation. Hydrogen bond interactions between the peptide (yellow carbon coloured stick) and PfSUB1 are indicated by dashed lines. (B) Root-mean square fluctuations (RMSF) about the mean position of the peptide backbone atoms. For RMSF calculation, overall translational and rotational motions were removed with respect to the Cα atoms of the PfSUB1 structure. (C) Scheme of hydrogen bonds formed during the MD simulation between PfSUB1 and peptide backbone and side-chain atoms. The peptide orientation is shown from N- to C-terminus left-to-right, and thus is opposite to the orientation shown in (A). Very strong hydrogen bonds (occupancy of 80–100%) are boxed in red, strong hydrogen bonds (occupancy of 60–80%) are in orange, and weaker hydrogen bonds (occupancy of 40–60%) are shown in green. Occupancy values were obtained over the course of the entire 50 ns of simulation time. Repeating the analysis for the first and second half of the trajectory resulted in the same hydrogen bond pattern.
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.

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