Isopeptide bond

[8] The biostructural roles of isopeptide bonds include blood clotting[9] (for wound healing), extracellular matrix upkeep,[10] the apoptosis pathway,[10] modifying micro-tubules,[11] and forming pathogenic pili[12] in bacteria.

Yet in case of RING finger domain containing that use coordination bonds with Zinc ions to stabilize their structures, they act more to direct the reaction.

[8] Thus while the internal mechanisms differ such as how proteins participate in the transfer chain, the general chemical aspects such as using thioesters and specific ligases for targeting remain the same.

However, the uniformity that exists in the ubiquitin’s case is not so here, as there are numerous different enzymes all performing the reaction of forming the isopeptide bond.

The general reaction performed by sortases involves using its own brand of the ‘catalytic triad’: i.e. using histidine, arginine, and cysteine for the reactive mechanism.

His and Arg act to help create the reactive environment, and Cys once again acts as the reaction center by using a thioester help hold a carboxyl group until the amine of a Lysine can perform a nucleophilic attack to transfer the protein and form the isopeptide bond.

[17] Then the thioester is resolved by the transfer of the peptide to a primary amine, and this generally has a very high specificity, which is seen in the example of B. cereus where the sortase D enzyme helps to polymerize the BcpA protein via two recognition signals, the LPXTG as the cleavage and thioester forming point, and the YPKN site which acts as the recognition signal as where the isopeptide will form.

The roles of these residues are analogous or the same as the previously described Sortases, in that His and Asp play a supporting role in interacting with the target residue, while the Cys forms a thioester with a carboxyl group for a later nucleophilic attack by a primary amine, in this case due to interest that of Lysine.

[21] It has also been shown to have some specificity as to which target Lysine it transfers the protein to, as in the case of Factor XIII, where the adjacent residue to the Lys decides whether the reaction will occur.

Another case of an isopeptide linking enzyme for structural purposes is the actin cross-linking domain (ACD) of the MARTX toxin protein generated by V. cholerae.

While it has been shown that the ACD when performing the catalysis uses magnesium and ATP for the formation of the cross-links the specifics of the mechanism are uncertain.

Both modifications are similar in the sense they are repeating stretches of the same amino acid fused to the side chain carboxyl group of glutamate at the c-terminal region of the MT.

[22] Though again there is a lack of clarity in regard to the enzymatic chemistry, there is still valuable insight in the formation of isopeptide bonds using the R-group carboxyl of Glu in conjunction with the N-terminal amino of the modifying peptides.

Spontaneous isopeptide bond formation has been exploited in the development a peptide tag called SpyTag.

SpyTag can spontaneously and irreversibly react with its binding partner (a protein termed SpyCatcher) through a covalent isopeptide bond.