Coagulation Factor XIIIa Inhibitor Tridegin: On the Role of Disulfide Bonds for Folding, Stability, and Function

Abstract

Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the fifteen possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogs using a targeted protecting-group strategy to investigate the impact of the C 19 -C 25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogs was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild type tridegin and the analogs as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C 19 -C 25 bond on conformational stability and binding mode. The strategy of

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure–Activity Relationships

“…To elucidate the structure of the two-disulfide-bonded isomer B [C19S,C25S] ( Figure 1 a) [ 29 ], two segments derived from the peptide sequence were synthesized for NMR measurements. The reason for dividing the tridegin isomer into an N-terminal and a C-terminal part was the size of the 66mer peptide and the high number of proline residues within the sequence, which complicate the assignment of the NMR signals and hamper structure calculation.…”

“…Therefore, a C-terminal fragment (amino acids 38–66) already described by Böhm et al [ 31 ] and a new N-terminal fragment (amino acids 1–37) with the disulfide bonds Cys5–Cys37 and Cys17–Cys31 were produced ( Supplementary Table S1, Supplementary Figure S1 ). For the synthesis of the latter fragment, a solid-phase peptide synthesis using the Fmoc-strategy with acetamidomethyl (Acm)- and trityl (Trt) -protecting groups (Cys5, Cys37 and Cys17, Cys31, respectively) and a subsequent stepwise oxidation in solution was conducted, similar to the strategy used for the earlier described two-disulfide-bonded tridegin analogues [ 29 ]. The stepwise oxidation process was performed in a one-pot reaction with iodine (oxidizing agent), whereby the former Trt-protected cysteines, were deprotected during the cleavage of the peptide from the solid support and initially linked, followed by the cleavage of the Acm-protected cysteines ( Figure 1 b).…”

“…Tridegin belongs to the class of cysteine-rich peptides due to its 6 cysteines within the amino acid sequence. The natural disulfide bond connectivity of tridegin could not be elucidated so far [ 28 , 29 , 30 , 31 ], which is due to the fact that a peptide with 6 cysteines has the ability to form 15 different three-disulfide-bonded isomers. So far, final conclusions have not been drawn concerning the folding of tridegin to the bioactive conformation and whether it is folded in a BPTI-like (Bovine pancreatic trypsin inhibitor) fashion, according to which one isomer is preferentially formed, or in a hirudin-like manner, where a maximum of 15 possible three-disulfide-bonded isomers are formed [ 32 , 33 ].…”

“…Therein, three of the 15 possible three-disulfide-bonded isomers were identified, which all shared the disulfide bond between Cys19–Cys25 and possessed similar activities toward FXIIIa [ 28 , 30 ]. In addition to the three-disulfide-bonded tridegin isomers, several variants lacking the disulfide bond between Cys19–Cys25 were synthesized in a following approach in order to determine the significance of this bond for their inhibitory potential against FXIIIa [ 29 ]. The results revealed that the activity against FXIIIa was not lost, despite the missing disulfide bond.…”

“…The results revealed that the activity against FXIIIa was not lost, despite the missing disulfide bond. Due to the unchanged activity and the improved synthetic outcome, the two-disulfide-bonded tridegin analogues, in particular the isomer B [C19S,C25S] with the disulfide bonds Cys5–Cys37 and Cys17–Cys31, are used as a lead structure for further research on tridegin and its influence on FXIIIa [ 29 ].…”