“…49,50 The scaffold of the peptides possibly confers an advantage in competitive binding over other bigger protease inhibitors, since the smaller size of these molecules could maximize interactions with the trypsin-like active sites. 51 The complementary analysis of enzymatic kinetic assays confirmed the competitive-inhibition model for GORE1 and GORE2 because both peptides fit the specific model assessed. The ability of GORE1 and GORE2 to inhibit trypsins of A. gemmatalis in vitro and the low inhibitory constants (K i ) obtained for the interaction indicate that these molecules are strong candidates to be used in the control of this insect pest.…”
Section: Discussionmentioning
confidence: 57%
“…The interaction pattern with S1 residues indicates a competitive inhibition model because they bind at the same spot as the natural substrate of trypsin‐like proteases 49,50 . The scaffold of the peptides possibly confers an advantage in competitive binding over other bigger protease inhibitors, since the smaller size of these molecules could maximize interactions with the trypsin‐like active sites 51 …”
“…49,50 The scaffold of the peptides possibly confers an advantage in competitive binding over other bigger protease inhibitors, since the smaller size of these molecules could maximize interactions with the trypsin-like active sites. 51 The complementary analysis of enzymatic kinetic assays confirmed the competitive-inhibition model for GORE1 and GORE2 because both peptides fit the specific model assessed. The ability of GORE1 and GORE2 to inhibit trypsins of A. gemmatalis in vitro and the low inhibitory constants (K i ) obtained for the interaction indicate that these molecules are strong candidates to be used in the control of this insect pest.…”
Section: Discussionmentioning
confidence: 57%
“…The interaction pattern with S1 residues indicates a competitive inhibition model because they bind at the same spot as the natural substrate of trypsin‐like proteases 49,50 . The scaffold of the peptides possibly confers an advantage in competitive binding over other bigger protease inhibitors, since the smaller size of these molecules could maximize interactions with the trypsin‐like active sites 51 …”
“…This may be due to the fact that SKTI is a protein containing 181 amino acid residues (Song & Suh, 1998) and thus contains cleavage sites for other enzymes that could be present in the midgut of A. gemmatalis and cause inactivation of the molecules by proteolysis. On the other hand, tripeptides exhibit longer retention and high stability in the insect gut, as shown elsewhere (Saikhedkar et al, 2018). The mechanism of disarming PIs by proteolytic cleavage is present in other insects, such as Otiorynchus sulcatus against oryzacystatin II (Michaud et al, 1995), Phaedon cochleariae against oryzacystatin I and Bowman-Birk inhibitor (Girard et al, 1998), Helicoverpa armigera against chickpea trypsin inhibitor (Giri et al, 1998), Spodoptera exigua against potato PI2 (Jongsma et al, 1995), and Plutella xylostella against MTI-2 ( Yang et al, 2009).…”
Insects overcome the action of natural protease inhibitors (PIs) due to evolutionary adaptations through endogenous proteolysis and reprogramming proteases. Insect adaptations complicate the formulation of IP‐based crop protection products. However, small peptides designed based on the active site of enzymes have shown promising results that could change this scenario. GORE1 and GORE2 are designed tripeptides that reduce the survival of Anticarsia gemmatalis when ingested orally. In this article, the stability and ability of the peptides to bind trypsin‐like enzymes of A. gemmatalis were evaluated by molecular dynamics (MD) simulations. The ability of the peptides to inhibit trypsin‐like enzymes in vivo was compared with the SKTI protein by feeding A. gemmatalis larvae at different concentrations, followed by an inhibition persistence assay. During the MD simulation of enzyme–ligand complexes, both peptides showed a small variation of root‐mean‐square deviation and root‐mean‐square fluctuation, suggesting that these molecules reach equilibrium when forming a complex with the trypsin‐like enzyme. Furthermore, both peptides form hydrogen bonds with substrate recognition sites of A. gemmatalis trypsin‐like enzyme, with GORE2 having more interactions than GORE1. Larvae of A. gemmatalis exposed to the peptides and SKTI showed a similar reduction in proteolytic activity, but the persistence of inhibition of trypsin‐like enzyme was longer in peptide‐fed insects. Despite their size, the peptides exhibit important active and substrate binding site interactions, stability during complex formation, and steadiness effects in vivo. The results provide fundamental information for the development of mimetic molecules and help in decision‐making for the selection of delivery methods for larger‐scale experiments regarding similar molecules.
“…6 a). It is reported that P2 Pro plays an essential role in determining the potency and specificity of the RCL [ 18 ]. Interestingly, Ser is rarely found at this position despite being structurally similar to Thr.…”
Section: Utility and Discussionmentioning
confidence: 99%
“…Each IRD contains a tripeptide loop called the reactive center loop (RCL), which provides target specificity to the Pin-II type PIs [ 17 ]. RCL is the primary interaction site for target serine protease and functions as an inhibitory tripeptide independent of the native IRD scaffold [ 18 ]. Fig.…”
Background
Serine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases.
Results
We have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family.
Conclusion
PINIR (https://pinir.ncl.res.in) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.
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