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 2.1Å resolution. The structure showed the expected reformation of the β-sandwich domain seen in the parental streptococcal adhesin, but flanking sequences at both 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 ultrastable molecular interaction.

Keywords: NE-CAT; Northeastern Collaborative Access Team; PEG; Streptococcus pyogenes; TEV; X-ray crystallography; bionanotechnology; cross-link; polyethylene glycol; synthetic biology; tobacco etch virus.

Fig. 1

Reconstitution of SpyTag/SpyCatcher complex. (a) Chemistry of isopeptide bond formation between the reactive Asp of SpyTag with Lys of SpyCatcher. (b) Gel analysis of reaction between SpyTag and SpyCatcher or SpyCatcherΔN1. Tag and protein, both at 50 μM in PBS, were incubated at room temperature for one hour before boiling in SDS-loading buffer. The samples were analyzed by SDS-PAGE and Coomassie staining. (c) Ribbon diagram of the SpyTag/SpyCatcher crystal structure. SpyTag is colored green and SpyCatcher is blue. The residues involved in the isopeptide are shown as sticks, with carbon atoms in gray. A second view of the structure is shown after 90° rotation.

Fig. 2

Structural contacts between SpyTag and SpyCatcher. (a) Electron density confirmation of the isopeptide bond between SpyCatcher and SpyTag. Residues of the reactive triad are shown in stick format with the 2Fo-Fc map contoured at 1 σ overlaid. (b) Stereo diagram showing the interface between SpyCatcher and SpyTag. SpyCatcher is shown as blue ribbons and the residues interacting with SpyTag are shown as sticks, with carbon atoms in gray. SpyTag is shown in green stick representation with its residues labeled. (c) Contacts between the tail of SpyTag (green) and SpyCatcherΔN1 (cyan). Putative hydrogen bonds are shown as magenta dotted lines. Terminal residues of SpyTag and contacting residues of SpyCatcher are labeled.

Fig. 3

Comparison between SpyTag/SpyCatcher and the intact CnaB2 domain. (a) Protein backbones are shown side-by-side in ribbon format, with SpyCatcher in blue and SpyTag in green. CnaB2 is shown in blue, except regions that are resolved in the CnaB2 structure but disordered in the SpyTag/SpyCatcher structure are shown in red. The region corresponding to SpyTag in CnaB2 is highlighted in green. (b) Secondary structure diagram for CnaB2 and SpyTag/SpyCatcher. β-strands are represented as arrows. Disordered peptides are shown as dashed lines. The ordered segments in CnaB2 that are invisible in SpyTag/SpyCatcher are drawn in red. The isopeptide is colored gray. SpyTag and the corresponding region in CnaB2 are colored green. (c) Comparison of putative main-chain hydrogen bonding to the C-terminal strand of CnaB2 (left) or SpyTag (right), shown in green stick format, with hydrogen bonds as dotted red lines. Residues from the rest of CnaB2 or SpyCatcher are shown in cyan line format.

Fig. 4

Testing reaction rates of structure-based mutants of SpyCatcher. (a) Sequence alignment of the N-terminal and C-terminal regions of the SpyCatcher deletion mutants, compared to the parental CnaB2, SpyCatcher and SpyTag sequences. All the internal regions of the mutants are identical to SpyCatcher. Red and blue arrows correspond to β-strands marked in Fig. 3b. Red boxes indicate deleted regions in SpyCatcher mutants. SpyTag residues that are identical to the C-terminal region of CnaB2 are boxed in green. (b) Quantification of SpyCatcher variant reaction with SpyTag-MBP (mean of triplicate ± 1 s.d. from technical replicates). Each partner at 10 μM was incubated at 25°C in 40 mM Na₂HPO₄ with 20 mM citric acid pH 7.0 for 1 or 10 min, before adding SDS-loading buffer, heating at 95°C for 7 min, and analysis on SDS-PAGE with Coomassie staining before gel densitometry, as previously. Reconstitution was determined as: 100× the band intensity of the covalent adduct, divided by the sum of intensities of the covalent adduct and SpyTag-MBP and the SpyCatcher variant.