Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin.
Keywords: BANA (N(α)-Benzoyl-DL-arginine β-naphthylamide) (PubChem CID: 102503); Cysteine proteinase inhibitor; Dimerization; E-64 (trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane) (PubChem CID: 439487); Inhibitory activity regulation; Papain; Poaceae; Protein modelling; Triticale (x Triticosecale); Z-Ala-Ala-Asn-AMC) (PubChem CID: 90470169); p-dimethylamino-cinnamaldehyde (PubChem CID: 5284506).
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