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The role of conserved residues of chagasin in the inhibition of cysteine peptidases.

dos Reis FC, Smith BO, Santos CC, Costa TF, Scharfstein J, Coombs GH, Mottram JC, Lima AP - FEBS Lett. (2008)

Bottom Line: A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi.A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases.These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil.

ABSTRACT
We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

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Chagasin residues targeted for mutagenesis. (A) Sequence alignment of chagasin (AJ299433), L. major ICP (AJ548878), L. mexicana ICP (AJ548776) and T. brucei ICP (AJ548777). The secondary structure is assigned on top and the conserved motifs are marked in bold. Conserved residues in loops L2, L4 and L6 are coloured blue, magenta, and green. The residues selected for site-directed mutagenesis are underlined. (B) Detail of the modeled interface between chagasin (beige) and cruzain (grey). Chagasin loops L2, L4 and L6 are coloured blue, magenta and green, respectively. Residues responsible for direct peptidase recognition and the peptidase’s catalytic triad are shown as sticks, and those mutated in this study are labeled and highlighted as fatter sticks.
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fig1: Chagasin residues targeted for mutagenesis. (A) Sequence alignment of chagasin (AJ299433), L. major ICP (AJ548878), L. mexicana ICP (AJ548776) and T. brucei ICP (AJ548777). The secondary structure is assigned on top and the conserved motifs are marked in bold. Conserved residues in loops L2, L4 and L6 are coloured blue, magenta, and green. The residues selected for site-directed mutagenesis are underlined. (B) Detail of the modeled interface between chagasin (beige) and cruzain (grey). Chagasin loops L2, L4 and L6 are coloured blue, magenta and green, respectively. Residues responsible for direct peptidase recognition and the peptidase’s catalytic triad are shown as sticks, and those mutated in this study are labeled and highlighted as fatter sticks.

Mentions: We used the findings from the studies of chagasin docking to cruzipain and chemical shift perturbation as a base to guide the site-directed mutagenesis [6,8]. The solution structure of chagasin suggested that the spatial arrangement of Loop2 (L2) might be influenced by the conserved Y89 residue, which is present on the β7 strand in close proximity to L2, and protrudes towards S28 [6]. Thus we selected this residue and also residues P30, T31, T32, composing the conserved NPTTG motif (L2), and the conserved W93 (L6) as targets for amino acid replacement in chagasin variants (Fig. 1A). The relative position of the residues selected for mutagenesis at the inhibitor–peptidase interface is shown in Fig. 1B. The relative importance of the side chains of T31 and T32 was investigated by studying variants bearing substitutions to charged, apolar and hydrophobic residues. We also addressed the importance of the length of L2 by the generation of a mutant devoid of residues T31 and T32 (see Table 1). All variants were generated using similar methods and SDS–PAGE of the purified proteins under reducing conditions showed that all variants had mobilities similar to that of chagasin (data not shown).


The role of conserved residues of chagasin in the inhibition of cysteine peptidases.

dos Reis FC, Smith BO, Santos CC, Costa TF, Scharfstein J, Coombs GH, Mottram JC, Lima AP - FEBS Lett. (2008)

Chagasin residues targeted for mutagenesis. (A) Sequence alignment of chagasin (AJ299433), L. major ICP (AJ548878), L. mexicana ICP (AJ548776) and T. brucei ICP (AJ548777). The secondary structure is assigned on top and the conserved motifs are marked in bold. Conserved residues in loops L2, L4 and L6 are coloured blue, magenta, and green. The residues selected for site-directed mutagenesis are underlined. (B) Detail of the modeled interface between chagasin (beige) and cruzain (grey). Chagasin loops L2, L4 and L6 are coloured blue, magenta and green, respectively. Residues responsible for direct peptidase recognition and the peptidase’s catalytic triad are shown as sticks, and those mutated in this study are labeled and highlighted as fatter sticks.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2607524&req=5

fig1: Chagasin residues targeted for mutagenesis. (A) Sequence alignment of chagasin (AJ299433), L. major ICP (AJ548878), L. mexicana ICP (AJ548776) and T. brucei ICP (AJ548777). The secondary structure is assigned on top and the conserved motifs are marked in bold. Conserved residues in loops L2, L4 and L6 are coloured blue, magenta, and green. The residues selected for site-directed mutagenesis are underlined. (B) Detail of the modeled interface between chagasin (beige) and cruzain (grey). Chagasin loops L2, L4 and L6 are coloured blue, magenta and green, respectively. Residues responsible for direct peptidase recognition and the peptidase’s catalytic triad are shown as sticks, and those mutated in this study are labeled and highlighted as fatter sticks.
Mentions: We used the findings from the studies of chagasin docking to cruzipain and chemical shift perturbation as a base to guide the site-directed mutagenesis [6,8]. The solution structure of chagasin suggested that the spatial arrangement of Loop2 (L2) might be influenced by the conserved Y89 residue, which is present on the β7 strand in close proximity to L2, and protrudes towards S28 [6]. Thus we selected this residue and also residues P30, T31, T32, composing the conserved NPTTG motif (L2), and the conserved W93 (L6) as targets for amino acid replacement in chagasin variants (Fig. 1A). The relative position of the residues selected for mutagenesis at the inhibitor–peptidase interface is shown in Fig. 1B. The relative importance of the side chains of T31 and T32 was investigated by studying variants bearing substitutions to charged, apolar and hydrophobic residues. We also addressed the importance of the length of L2 by the generation of a mutant devoid of residues T31 and T32 (see Table 1). All variants were generated using similar methods and SDS–PAGE of the purified proteins under reducing conditions showed that all variants had mobilities similar to that of chagasin (data not shown).

Bottom Line: A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi.A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases.These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Bloco G, C.C.S., Cidade Universitária, Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil.

ABSTRACT
We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the K(i) for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

Show MeSH