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Volume 31, Issue 9
  • ISSN: 0929-8665
  • E-ISSN: 1875-5305

Abstract

Background

Different inhibitor families have their own conserved three-dimensional structures, but how these structures determine whether a protein can become an inhibitor is still unknown. The buckwheat trypsin inhibitor (BTI) pertains to the Potato I type inhibitor family, which is a simple and essential bio-molecule that serves as a model for the investigation of protease-inhibitor interaction.

Objective

To study the effects of mutations at Trp10 and Ile25 in the hydrophobic cavity (scaffold) of rBTI on its inhibitory activity and stability.

Methods

Site-directed mutagenesis and molecular modeling were performed using the sequence of BTI. The hydrogen bonds formed by all amino acids and conformational differences of Trp53 were analyzed in the tertiary structures of rBTI and mutants.

Results

Mutant rBTI-W10A almost completely lost its inhibitory activity (retaining 10%), while rBTI-I25A retained about 50% of its inhibitory activity. Both rBTI-W10A and rBTI-I25A could be degraded by trypsin. The hydrogen bond analysis results showed that mutating Trp10 or Ile25 weakened the specific cohesion interactions in the hydrophobic core of rBTI, disrupting the tight hydrogen bond network in the cavity. This further led to difficulty in maintaining the binding loop conformation, ultimately causing the Trp53 to undergo conformational changes. It was also difficult for residues in the mutants to form hydrogen bonds with amino acids in bovine trypsin; thus, the mutants could not stably bind to trypsin.

Conclusion

Our findings suggest that the hydrophobic core is also an important factor in the maintenance of inhibitory activity and stability of rBTI.

© 2024 Bentham Science Publishers
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2024-09-25
2024-12-23

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A Conserved Tryptophan (Trp10) at the Hydrophobic Core Modulates the Stability and Inhibitory Activity of Potato I Type Inhibitors
PPL 31, 736 (2024); https://doi.org/10.2174/0109298665333930240905111039
/content/journals/ppl/10.2174/0109298665333930240905111039
/content/journals/ppl/10.2174/0109298665333930240905111039
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Supplements

Table for the content of the secondary structure of rBTI and mutants. Tables shows the Ramachandran plot statistics of rBTI-W10A and rBTI-I25A. Supplementary material is available on the publisher’s website along with the published article.

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  • Article Type:     Research Article
Keyword(s): bovine trypsin; hydrogen bonds; inhibitory activity; Potato I type inhibitor; stability; tryptophan

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