Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Guerra, Yasel; Valiente, Pedro A.; Pons, Tirso; Berry, Colin; Rudino‐Pinera, E.

doi:10.1016/j.jsb.2016.06.014

Cited by 7 publications

(2 citation statements)

References 80 publications

(115 reference statements)

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“…The conformational similarity between the β5–β6 loop of API and potato inhibitors API‐11, API‐3, and PSPI, was previously reported by us (Guerra et al, 2016); but, to the best of our knowledge, there are no previous reports of the conformational similarity with inhibitor STI β5–β6 loop. This is interesting considering that STI can bind trypsin and chymotrypsin simultaneously, and amino acids M84 and L85 have been proposed as potential P1 and P1′ positions in the second binding site (Bösterling & Quast, 1981).…”

Section: Discussionsupporting

confidence: 76%

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

et al. 2023

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The Kunitz‐Soybean Trypsin Inhibitor (Kunitz‐STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz‐STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz‐STI inhibitors to characterize serine protease‐binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease‐binding loops and the inhibition mechanism, their location in the β‐trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4–β5 loop, whereas serine protease‐binding loops in inhibitors from other plants lie mainly in the β5–β6 and β9–β10 loops. In addition, we found that the β5–β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz‐STI inhibitors are canonical inhibitors and proteins with protease‐binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease‐binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

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Section: Discussionsupporting

confidence: 76%

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

et al. 2023

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“…Encouraged that SBTI is stable in gastrointestinal conditions and heat-resilient, we explored the evolvability of this candidate scaffold protein, to generate what we term a gastrobody. β-trefoil fold proteins tend to share similar tertiary structure, but have low sequence identity, except for some inward-facing hydrophobic residues 40 . Structural homology despite a lack of sequence conservation suggests that the β-trefoil fold may be amenable to directed evolution and engineering.…”

Section: Resultsmentioning

confidence: 99%

Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid

2021

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Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

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Kunitz‐type trypsin inhibitor from durian (Durio zibethinus) employs a distinct loop for trypsin inhibition

Deetanya,

Limsardsanakij,

Sabat

et al. 2024

Protein Science

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Kunitz‐type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz‐type trypsin inhibitors as identified by N‐terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI‐4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI‐4 utilized a novel tryptophan‐containing β1‐β2 loop to bind trypsin. Site‐direct mutagenesis confirmed the inhibitory role of this loop. DzTI‐4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum‐free conditions. DzTI‐4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz‐type trypsin inhibitors and provides clues for further application of these inhibitors.

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Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Cited by 7 publications

References 80 publications

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid

Kunitz‐type trypsin inhibitor from durian (Durio zibethinus) employs a distinct loop for trypsin inhibition

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