Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag

Abstract

Peptide tagging is a key strategy for observing and isolating proteins. However, the interactions of proteins with peptides are nearly all rapidly reversible. Proteins tagged with the peptide SpyTag form an irreversible covalent bond to the SpyCatcher protein via a spontaneous isopeptide linkage, thereby offering a genetically-encoded way to create peptide interactions that resist force and harsh conditions. Here, we determined the crystal structure of the reconstituted covalent complex of SpyTag and SpyCatcher at 2.1 Å resolution. The structure showed the expected reformation of the β-sandwich domain seen in the parental streptococcal adhesin, but flanking sequences at both the N- and C-termini of SpyCatcher were disordered. In addition, only 10 out of 13 amino acids of the SpyTag peptide were observed to interact with SpyCatcher, pointing to specific contacts important for rapid split protein reconstitution. Based on these structural insights, we expressed a range of SpyCatcher variants and identified a minimized SpyCatcher, 32 residues shorter, that maintained rapid reaction with SpyTag. Together, these results give insight into split protein β-strand complementation and enhance a distinct approach to ultra-stable molecular interaction.

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

“…The sequence VPT‐ was used thereafter at the N terminus, while the C terminus was randomized based on this lead. After rounds of phage library screening, the enriched hits CLib1‐10 are shown (Figure 1 c), with their position on the parent structure indicated (Figure 1 d) 6. Of these variants, CLib1 (identified in two separate clones, also as CLib9) was fastest for reaction with SpyCatcher and preserved the YK pair at residues 9–10 of WT SpyTag.…”

Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry

“…1C). A recent structural study shows that SpyTag and SpyCatcher form a compact β-sandwich structure, in which SpyCatcher interacts with only one side of SpyTag (17). Multiple interactions are involved in formation of the complex, including strong hydrophobic interactions between side chains and an extensive hydrogen bond network, in addition to the covalent isopeptide bond (17).…”

“…Multiple interactions are involved in formation of the complex, including strong hydrophobic interactions between side chains and an extensive hydrogen bond network, in addition to the covalent isopeptide bond (17). The binding site of SpyCatcher is solvent exposed and allows fast complementation of SpyTag and SpyCatcher (17). Mutation of Asp-117 to Ala in SpyTag has been shown to abolish covalent isopeptide bond formation but to maintain noncovalent interactions that drive formation of the complex with a K d of 0.2 μM (12), suggesting a relatively strong protein-peptide interaction (18)(19)(20).…”

Nouvelles stratégies d’innovations vaccinales et leurs application en médecine vétérinaire

“…Ces deux briques s'auto-assemblent spontanément pour former un pont isopeptidique de manière quasi-irréversible. La liaison est stable entre +4 et 37°C, dans une gamme de pH de 5 à 8, dans toutes sortes de milieux et même en présence de certains détergents (Li et al 2014;Reddington & Howarth, 2015). En recouvrant la surface de nanoparticules avec l'une des molécules de la paire SpyTag / SpyCatcher, et en attachant l'autre molécule de la paire sur les antigènes d'intérêt, il suffira de mélanger les deux pour créer un vaccin extrêmement stable, et ciblant directement les cellules dendritiques, qui sont programmées pour détecter de telles structures particulaires répétitives (figure 1).…”