High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector<i>Aedes aegypti</i>

Torquato, Ricardo J.S.; Lu, Stephen; Martins, N.H.; Tanaka, Aparecida S.; Pereira, Pedro José Barbosa

doi:10.1107/s2053230x17010007

Cited by 5 publications

(4 citation statements)

References 46 publications

(50 reference statements)

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“…AaTI is a competitive inhibitor of plasmin ( K i = 3.8 nM) but it does not inhibit kallikreins, FXa or FXIIa [ 17 ], and only weakly inhibits thrombin ( K i = 320 nM) [ 18 ]. AaTI has a similar structure to that of Inf4 [ 19 ], and with only a 3-residue difference in the reactive-site loop sequence. The reactive-site loop sequence makes most-if not all-of the direct contacts with the protease and is thought to be the sole factor that govern the inhibitor specificity.…”

Section: Introductionmentioning

confidence: 99%

Scaffold stability and P14′ residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4

et al. 2022

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Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively-charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most—if not all—of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only 3 residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14’ position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.

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Section: Introductionmentioning

confidence: 99%

Scaffold stability and P14′ residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4

et al. 2022

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“…The reactive-site loop is located between Cys II and Cys III. 10 Structures of complexes formed between Kazal-type inhibitors and serine proteases are also available. For instance, Inf1 and Inf4 in complex with trypsin 9 ; LDTI in complex with trypsin 15,16 ; rhodniin, from the Chagas' disease vector Rhodnius prolixus, in complex with thrombin 17 ; and greglin, from ovaries of the locust Schistocerca gregaria, in complex with subtilisin.…”

Section: Introductionmentioning

confidence: 99%

“…The 3D structures of many Kazal‐type inhibitors are known, including Inf1 and Inf4 9 ; AaTI 10 ; leech‐derived tryptase inhibitor (LDTI) 11 ; Anemonia elastase inhibitor 12 ; lympho‐epithelial Kazal‐type inhibitor 13 ; dipetalin (Dip‐I) from Mexican predatory, blood‐sucking insect Dipetalogaster maximus 14 ; and CmPI‐II 5 . Kazal‐type inhibitors have a typical fold of 2 or 3 α‐helices and a short 3‐stranded antiparallel β‐sheet 11 .…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of Aedes aegypti trypsin inhibitor in complex with μ‐plasmin reveals role for scaffold stability in Kazal‐type serine protease inhibitor

et al. 2021

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Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.

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Exploring oak processionary caterpillar induced lepidopterism (Part 1): unveiling molecular insights through transcriptomics and proteomics

Seldeslachts,

Maurstad,

Øyen

et al. 2024

Cell. Mol. Life Sci.

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Lepidopterism, a skin inflammation condition caused by direct or airborne exposure to irritating hairs (setae) from processionary caterpillars, is becoming a significant public health concern. Recent outbreaks of the oak processionary caterpillar (Thaumetopoea processionea) have caused noteworthy health and economic consequences, with a rising frequency expected in the future, exacerbated by global warming promoting the survival of the caterpillar. Current medical treatments focus on symptom relief due to the lack of an effective therapy. While the source is known, understanding the precise causes of symptoms remain incomplete understood. In this study, we employed an advanced method to extract venom from the setae and identify the venom components through high-quality de novo transcriptomics, venom proteomics, and bioinformatic analysis. A total of 171 venom components were identified, including allergens, odorant binding proteins, small peptides, enzymes, enzyme inhibitors, and chitin biosynthesis products, potentially responsible for inflammatory and allergic reactions. This work presents the first comprehensive proteotranscriptomic database of T. processionea, contributing to understanding the complexity of lepidopterism. Furthermore, these findings hold promise for advancing therapeutic approaches to mitigate the global health impact of T. processionea and related caterpillars.

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High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vectorAedes aegypti

Cited by 5 publications

References 46 publications

Scaffold stability and P14′ residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4

Scaffold stability and P14′ residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4

Crystal structure of Aedes aegypti trypsin inhibitor in complex with μ‐plasmin reveals role for scaffold stability in Kazal‐type serine protease inhibitor

Exploring oak processionary caterpillar induced lepidopterism (Part 1): unveiling molecular insights through transcriptomics and proteomics

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