Abstract
Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of “furan-armed” α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains.
Highlights
Peptide–protein and protein–protein interactions play key roles in biological processes
Introduction of the furan moiety for cross-linking involves the precursor unnatural amino acid Orn, which is coupled with 2-furanpropionic acid (Fur) through the amino group of the Orn side chain placed at position 13 on the ECoil (Figure 3A, resulting in the sequence ECoil-OrnFur-13)
The KP-10-GPR54 homology model as well as the crystal structure of Actin-Tβ4 (4PL7) (Figure 2) indicate that besides Lys, other side-chain amino acids can be located in close proximity to the activated furan moiety
Summary
Peptide–protein and protein–protein interactions play key roles in biological processes. A furan modified kisspeptin-10 peptide ligand could be covalently cross-linked to its membrane receptor, GPR54, on live cancer cells with spontaneous oxidation of the furan moiety by endogenous ROS (Figure 1A) (Vannecke et al, 2017). We could demonstrate that upon incubation of live cells expressing the GPR54 receptor with the furan-modified peptide, a covalently cross-linked kisspeptin-10/GPR54 complex was formed as demonstrated by extensive analysis of the cell lysate by Western blotting (Vannecke et al, 2017).
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