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

Highlights

• SpyTag/SpyCatcher provides a unique covalent peptide-tagging system.

• The crystal structure of SpyTag/SpyCatcher was determined at 2.1 Å resolution.

• The structure predicts that the N- and C-termini of SpyCatcher are dispensable.

• Biochemical experiments confirm the structural predictions.

• The results lead to an optimized SpyTag/SpyCatcher system.

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 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.

Introduction

The tagging of proteins with peptides is one of the most widely used methods in protein detection, purification and immobilization [1], [2], [3]. In most cases, the peptide tag interacts reversibly, either with a protein, such as an antibody or a specific binding partner, or with a metal chelate. In contrast, the recently developed SpyTag/SpyCatcher tagging system involves the covalent attachment of a peptide tag to its cognate protein partner [4]. The system is based on the immunoglobulin-like collagen adhesion domain of Streptococcus pyogenes (CnaB2). CnaB2 contains an internal isopeptide bond [5] between amino acid residue Lys31 and residue Asp117 [6], [7], [8]. When CnaB2 is split into an N-terminal protein fragment containing Lys31 and a C-terminal peptide containing Asp117, the two fragments associate specifically and spontaneously form the isopeptide bond (Fig. 1a). A few modifications to the two binding partners made the reaction efficient both in vitro and in vivo. The modified peptide and protein fragment were named SpyTag and SpyCatcher, respectively [4].

The SpyTag/SpyCatcher system offers several advantages over other tagging approaches. SpyTag (13 amino acids) forms a high-affinity initial non-covalent complex with its protein partner SpyCatcher (116 amino acids). The two partners then react rapidly, forming the isopeptide bond, with a half-time of 74 s for partners at 10 μM [4]. The reaction can take place in diverse conditions and is relatively insensitive to pH and temperature changes. Due to the covalent nature of the isopeptide bond, the SpyTag/SpyCatcher complex forms irreversibly and is stable to boiling in SDS or to forces of thousands of piconewtons [4]. The SpyTag can be placed at the N-terminus, at the C-terminus and at internal positions of a protein [4], in contrast to covalent peptide labeling via split inteins [9], [10] or sortases [11]. Thus, the SpyTag/SpyCatcher system is potentially versatile and general. However, a better understanding of the interaction between the two partners is required to optimize the system.

Split proteins are an important and rapidly growing protein class, including split luciferase, fluorescent proteins, DNA polymerase and proteases. Split proteins give important insight into protein folding and are powerful tools for logical computation or for reporting on diverse cellular events [12]. However, there are very few studies of how different split proteins reconstitute to form the original fold [13], [14]. Here, we have analyzed the binding of SpyTag and SpyCatcher using X-ray crystallography and biochemical methods. The crystal structure of the SpyTag and SpyCatcher complex indicates that the N-terminal and C-terminal segments of SpyCatcher are dispensable for the interaction. Our biochemical and structural studies confirm that both termini could be deleted without a major effect on the structure or reaction rate. In addition, the crystal structure explains the effect of previously engineered point mutations on the reaction efficiency. Together, these results lead to an optimized and robust SpyTag/SpyCatcher system.