Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick

Abstract

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential, besides rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of BmTI-A was modeled based on the structure of its Sabellastarte magnifica homologue. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism data corroborated the secondary structure found by the homology modeling.Thermostability analysis confirmed the thermostability and the relation between the effects of the temperature in the inhibitor activity. The loss of activity observed was gradual, and, after 60 minutes of incubation at 90ºC the inhibitor lost it completely.

Kunitz-Type Peptides from Sea Anemones Protect Neuronal Cells against Parkinson’s Disease Inductors via Inhibition of ROS Production and ATP-Induced P2X7 Receptor Activation

“…This feature allows them to maintain their protease-inhibitory activity over a wide range of temperatures and pH values. Indeed, the Kunitz peptide SdPI from the scorpion Lychas mucronatus [58] and BmTI-A from tick Rhipicephalus microplus [59] were shown to be thermostable, while Bm-SPI51 from the cocoon of the silkworm Bombyx mori is both heat-and pH-resistant [60]. Based on the CD spectra and residual trypsin activity levels, we revealed that HCIQ2c1, HCIQ4c7, and HMIQ3c1 are thermostable peptides retaining the conformation of the active molecules up to 90-100 • C, whereas the trypsin-inhibitory activity of HCIQ1c9 is reduced by up to 70% in the temperature range of 60-100 • C, an observation that was confirmed by the CD spectrum.…”

“…Substrate hydrolysis was measured at 410 nm. The residual activity was calculated based on the results of three parallel experiments according to the following equation [60]: % = (1 − residual enzyme activity/enzyme activity without inhibitor) × 100…”

Novel proteinase inhibitor from the hemolymph of soybean pest Anticarsia gemmatalis (lepidóptera: Noctuidae): Structural modeling and enzymatic kinetic

“…Has been corroborated that coiled-coil regions are important because they are strictly related to the correct assembly of the protein's final and functional structure (Linding et al, 2003). Some studies suggest that these regions have been known as linkers, probes, and inhibitors of protein-protein interactions (Bomediano Camillo et al, 2021;Watkins et al, 2015).…”