Investigation of Serine‐Proteinase‐Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI‐1)

Abstract

Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.

Spliced analogues of trypsin inhibitor SFTI‐1 and their application for tracing proteolysis and delivery of cargos inside the cells

“…The incubation of such peptides with equimolar amounts of proteinase resulted in the synthesis of a peptide bond that gives monocyclic SFTI‐1 inhibitors (Figure ). By applying HPLC/MS and X‐ray analyses, we proved that, regardless of the extension at the C‐ terminus by Ser of the first chain or by Lys at the N‐ terminus of the second chain, spliced monocyclic SFTI‐1 was formed . Using O, we confirmed that, similarly to the mechanism of peptide splicing catalyzed by the proteasome, the splicing of SFTI‐1 analogues also follows a direct transpeptidation mechanism.…”

P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing

“…With respect to ultra-high-resolution data collection, the crystal structure of the sunflower trypsin inhibitor 1 (SFTI-1) (Karna et al, 2015) and the structures of open and closed states of Candida antarctica lipase B (Stauch et al, 2015) were determined at 0.91 Å resolution using X-rays at = 1.00 Å (12.4 keV) and = 0.826 Å (15.0 keV), respectively.…”

Linking inhibitor motions to proteolytic stability of sunflower trypsin inhibitor-1

“…[8][9][10][11] Moreover, the very small size, strong inhibitory and high hydrolysis stability 12,13 of SFTI-1 have made it an excellent scaffold 14 for protein engineering. [15][16][17][18][19][20] Sequences with completely novel function can be graed into the SFTI-1 framework, for engineering of radiopharmaceuticals, antimicrobials, proangiogenic compounds, and rheumatoid arthritis and autoimmune disease peptides. [21][22][23][24][25][26][27][28] SFTI-1 binds to trypsin in a substrate manner but can resist proteolysis for a quite long time.…”

“…Lack of either a disulde bond or cyclic backbone showed decreased proteinase resistance to trypsin and inhibitory activity. 6,13,20,[29][30][31][32] The studies on two monocyclic analogs of SFTI-1 showed that the one keeping head-to-tail cycle was remarkably faster hydrolyzed than the with a disulde bridge. 13 On top of it, the hydrogen-bond network also appears to be a crucial determinant of the function of SFTI-1.…”