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Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

Valdés JJ, Schwarz A, Cabeza de Vaca I, Calvo E, Pedra JH, Guallar V, Kotsyfakis M - PLoS ONE (2013)

Bottom Line: Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin.The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic. valdjj@gmail.com

ABSTRACT

Background: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions.

Results: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).

Conclusions: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

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A–B. Coarse grain docking of refined tryptogalinin model and tryptogalinin-trypsin complex.Top two panels (A) show the coarse grain binding against the RMSD for two tryptogalinin models, Tryp1: the last snapshot of a 62 ns equilibration, and Tryp2, the snapshot with best superimposition to TdPI (its complex with trypsin) (PDB: 2UUY). RMSD were obtained with respect to the superimposition of tryptogalinin to 2UUY. Bottom panels show the all-atom binding energy after clusterization of the coarse grain poses. A comparison between (B) the best all-atom model for tryptogalinin (yellow) and the complex TdPI-trypsin crystal structure (PDB: 2UUY) (red) depict significant binding similarities. Lys13 and 34, and Asp191 (green) are represented in ball and stick.
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pone-0062562-g007: A–B. Coarse grain docking of refined tryptogalinin model and tryptogalinin-trypsin complex.Top two panels (A) show the coarse grain binding against the RMSD for two tryptogalinin models, Tryp1: the last snapshot of a 62 ns equilibration, and Tryp2, the snapshot with best superimposition to TdPI (its complex with trypsin) (PDB: 2UUY). RMSD were obtained with respect to the superimposition of tryptogalinin to 2UUY. Bottom panels show the all-atom binding energy after clusterization of the coarse grain poses. A comparison between (B) the best all-atom model for tryptogalinin (yellow) and the complex TdPI-trypsin crystal structure (PDB: 2UUY) (red) depict significant binding similarities. Lys13 and 34, and Asp191 (green) are represented in ball and stick.

Mentions: To further test this hypothesis we proceeded by superimposing all the tryptogalinin MD snapshots to TdPI and found one structure with only 0.9Å RMSD (for the Lys all-atom RMSD). Then we used this structure (Tryp2), plus the equilibrated MD model (Tryp1; with a ∼4Å Lys-Asp RMSD), for the following round of protein-protein docking studies. This time, we used a biased docking approach based on the Basdevant et al. [43] coarse grain (CG) potential, a model with only 2–3 beads per residue that softens the steric repulsion and favors the surface contacts. The top two panels in Figure 7A shows 300,000 Monte Carlo steps for the CG exploration, where we biased tryptogalinin to the active site (see Materials and Methods section). Tryp2, the tryptogalinin MD conformation with better superimposition to TdPI, enters the active site reaching Lys13-Asp191 distances <4Å with a significant correlation between the CG binding energy and the RMSD. In agreement with the previous docking experiments, the equilibrated MD structure is not capable of entering the active site. From these CG results we clustered 100 poses where we imposed the distance between Lys13 and Asp191 to be <8Å, and refined them with all-atom models (see Materials and Methods section). The bottom two panels in Figure 7A show the all-atom binding energy. Clearly, Tryp2 produces again a significant correlation of the binding energy with the RMSD, using the 2UUY model as reference, indicating that both proteins bind similarly (as could be expected by their phylogenetic relation and similarities in protease inhibition – e.g., β-tryptase).


Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

Valdés JJ, Schwarz A, Cabeza de Vaca I, Calvo E, Pedra JH, Guallar V, Kotsyfakis M - PLoS ONE (2013)

A–B. Coarse grain docking of refined tryptogalinin model and tryptogalinin-trypsin complex.Top two panels (A) show the coarse grain binding against the RMSD for two tryptogalinin models, Tryp1: the last snapshot of a 62 ns equilibration, and Tryp2, the snapshot with best superimposition to TdPI (its complex with trypsin) (PDB: 2UUY). RMSD were obtained with respect to the superimposition of tryptogalinin to 2UUY. Bottom panels show the all-atom binding energy after clusterization of the coarse grain poses. A comparison between (B) the best all-atom model for tryptogalinin (yellow) and the complex TdPI-trypsin crystal structure (PDB: 2UUY) (red) depict significant binding similarities. Lys13 and 34, and Asp191 (green) are represented in ball and stick.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643938&req=5

pone-0062562-g007: A–B. Coarse grain docking of refined tryptogalinin model and tryptogalinin-trypsin complex.Top two panels (A) show the coarse grain binding against the RMSD for two tryptogalinin models, Tryp1: the last snapshot of a 62 ns equilibration, and Tryp2, the snapshot with best superimposition to TdPI (its complex with trypsin) (PDB: 2UUY). RMSD were obtained with respect to the superimposition of tryptogalinin to 2UUY. Bottom panels show the all-atom binding energy after clusterization of the coarse grain poses. A comparison between (B) the best all-atom model for tryptogalinin (yellow) and the complex TdPI-trypsin crystal structure (PDB: 2UUY) (red) depict significant binding similarities. Lys13 and 34, and Asp191 (green) are represented in ball and stick.
Mentions: To further test this hypothesis we proceeded by superimposing all the tryptogalinin MD snapshots to TdPI and found one structure with only 0.9Å RMSD (for the Lys all-atom RMSD). Then we used this structure (Tryp2), plus the equilibrated MD model (Tryp1; with a ∼4Å Lys-Asp RMSD), for the following round of protein-protein docking studies. This time, we used a biased docking approach based on the Basdevant et al. [43] coarse grain (CG) potential, a model with only 2–3 beads per residue that softens the steric repulsion and favors the surface contacts. The top two panels in Figure 7A shows 300,000 Monte Carlo steps for the CG exploration, where we biased tryptogalinin to the active site (see Materials and Methods section). Tryp2, the tryptogalinin MD conformation with better superimposition to TdPI, enters the active site reaching Lys13-Asp191 distances <4Å with a significant correlation between the CG binding energy and the RMSD. In agreement with the previous docking experiments, the equilibrated MD structure is not capable of entering the active site. From these CG results we clustered 100 poses where we imposed the distance between Lys13 and Asp191 to be <8Å, and refined them with all-atom models (see Materials and Methods section). The bottom two panels in Figure 7A show the all-atom binding energy. Clearly, Tryp2 produces again a significant correlation of the binding energy with the RMSD, using the 2UUY model as reference, indicating that both proteins bind similarly (as could be expected by their phylogenetic relation and similarities in protease inhibition – e.g., β-tryptase).

Bottom Line: Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin.The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic. valdjj@gmail.com

ABSTRACT

Background: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions.

Results: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).

Conclusions: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

Show MeSH