Spontaneous Intermolecular Amide Bond Formation between Side Chains for Irreversible Peptide Targeting

Abstract

Peptides and synthetic peptide-like molecules are powerful tools for analysis and control of biological function. [1][2][3] One major problem with the use of peptides is the instability of their interactions with biomolecules, with typically micromolar affinity relating to the limited accessible surface area 4,5 and the intrinsic flexibility of peptides. 6 However, appending a short peptide tag is the most common way to allow a protein of interest to be isolated or detected, giving minimum perturbation to protein function. 7,8 Here we have designed a way to bind a peptide tag irreversibly, by adapting a recently discovered feature of amino acid chemistry: the spontaneous formation of an amide bond between a Lys and an Asn side chain in the appropriate environment. [9][10][11] Amide linkages outside of the protein main chain are termed isopeptide bonds (Figure 1A). Isopeptide bonds are chemically stable and resistant to most proteases. 12,13 Enzymes such as transglutaminases catalyze isopeptide formation, stabilizing the extracellular matrix and strengthening blood clots, 12 but these enzymes are large and have low sequence specificity. 12 Recently, certain proteins were discovered to autocatalyze single-turnover isopeptide bond formation, yielding ultrathin viral capsid chain mail, 9 or the proteolytically stable pili of Gram-positive bacteria, 10,11 through nucleophilic attack of the ε-amino group from a Lys to the Cγ group of an Asn, promoted by a nearby Glu (Figure 1B). 9,10 To apply spontaneous isopeptide bond formation to direct new covalent peptide interactions, we dissected the major pilin protein Spy0128 from Streptococcus pyogenes 10 (Figure 1C) and explored whether the two fragments would covalently associate. Split proteins have successfully reconstituted in many cases, including enzymes and fluorescent proteins, 14 albeit through noncovalent interactions. Spy0128 was split at the final β-strand of the C-terminal domain, to give the fragment pilin-C (Spy0128 residues 18-299, with N-terminal His 6 ) and the isopeptag (Spy0128 residues 293-308: TDKDMTITFTNKKDAE). This placed the reactive Asn on the isopeptag and the reactive Lys on pilin-C. To enhance recombinant expression in E. coli, the isopeptag was genetically fused to the N-terminus of maltose binding protein (MBP). To test covalent reaction, we mixed isopeptag-MBP and pilin-C, each at 10 µM, and boiled the samples in SDS before SDS-PAGE (Figure 1D). A new product formed at ∼80 kDa, consistent with reaction between isopeptag-MBP and pilin-C. We verified amide bond formation between isopeptag-MBP and pilin-C by mass spectrometry, demonstrating the loss of NH 3 upon reaction (Figure 2A). Pilin-C K179A, lacking the reactive Lys, did not form a covalent complex with isopeptag-MBP, determined by SDS-PAGE (Figure 1D) and mass spectrometry (Figure S1). Also, pilin-C did not react with MBP fused to an alternative peptide containing four potentially reactive Asn residues (MBP-isopeptag-N, Figure 1D).Spy0128 contains another isopeptide bond in its N-terminal...

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

“…Like Spy0128, in CnaB2 the isopeptide is between the N-terminal and C-terminal β-strands, so that, if the isopeptide does not break, then the force on the protein will not cause any extension of the protein chain. Intermolecular reconstitution can be achieved with split Spy0128 but the partners react orders of magnitude slower, undergo side reactions, and the protein is undesirably large (29). The stability of the SpyTag interaction before the isopeptide bond forms may relate to the known mechanical strength of long parallel β-strands, where multiple hydrogen bonds have to break simultaneously (23).…”

Programmable polyproteams built using twin peptide superglues

“…In addition, it is preferable for modules to be modified with peptide tags rather than protein fusion domains (e.g., HaloTag or SNAP-tag) for minimal disruption to module function (22,23). For unbreakable linkage to a peptide, we previously developed the use of spontaneous isopeptide bond formation (24,25). SpyTag is a 13-amino-acid peptide that can be genetically fused to the protein of interest, and upon mixing with its protein partner SpyCatcher, an Asp of SpyTag forms a spontaneous isopeptide bond with a Lys of SpyCatcher (26).…”

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

“…SpyTag002 and SpyCatcher002 demonstrated rapid reaction under a wide range of buffers, temperatures, and pH values, and as N‐terminal or C‐terminal fusions. SpyTag002/SpyCatcher002 allowed specific covalent pulse‐labeling of surface proteins on living cells and represents the fastest currently available Tag/Catcher pair 2b, 12. In future work it will be important to test these new variants for challenging in vitro labeling, such as coupling antigens at high density on virus‐like particles for vaccination 13.…”