Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

Abstract

SpyTag is apeptide that forms aspontaneous amide bond with its protein partner SpyCatcher.T his protein superglue is ab roadly useful tool for molecular assembly,l ocking together biological building blocks efficiently and irreversibly in diverse architectures.W ei nitially developed SpyTag and SpyCatcher by rational design, through splitting ad omain from aG ram-positive bacterial adhesin. In this work, we established ap hage-displayp latform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under awide range of conditions and at either protein terminus.S pyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.Thousands of non-covalent protein-protein interactions mediate cellular function. However,e ngineering covalent interactions between protein partners brings ar ange of new opportunities for basic research and synthetic biology.[1] We have developed the use of spontaneous amide bond formation by peptide tags as asimple,specific, and genetically-encoded route to lock protein units together.[2] This technology, particularly the SpyTag/SpyCatcher pair, has been used in diverse applications including biomaterials,n ext-generation sequencing,e nzyme stabilization, and vaccine development. [1a, 3] Ak ey limitation has been relatively slow reaction at cellular expression levels.W ee stablished an evolutionary approach to achieve as econd-generation, faster-reacting version of this protein superglue.W et hen applied the enhanced properties for efficient and specific cell-surface functionalization, to investigate the outer-membrane dynamics of intimin, ap rotein relevant to human colonization by pathogenic bacteria.Since the SpyTag/SpyCatcher system is an unconventional approach to peptide interaction, it is likely that there are features of the interaction that cannot be predicted by rational design. Selection from phage libraries has been established for decades and the difficult thing is usually to detect weak interactions, [4] rather than the challenge of

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

“…To test whether SpyC could access and covalently react with the RLC‐Spy incorporated into filaments, SpyC (1 μM) was mixed with SMM RLC‐Spy filaments (0.5 μM, uncrosslinked) and the reaction time course was monitored by the appearance of a ∼35 kDa band by SDS gel electrophoresis (Figure c). As expected (Keeble et al, ; Zakeri et al, ), the reaction reached ∼90% completion within 20 min. Figure d shows that the filaments remained intact after this reaction occurred.…”

“…His 6 -Cys-SpyCatcher002 protein expression was as described in (Keeble et al, 2017). pDEST14-Cys-SpyCatcher002 was a gift from M. Howarth (Addgene plasmid # 102829).…”

“…To completely avoid QD interference with filament formation, we concluded that it was necessary to attach the QD to the RLC after filaments had already been formed. Our approach was to use the SpyTag002 (Spy) and SpyCatcher002 (SpyC) system (Keeble et al, 2017). We expressed RLC constructs with N-or C-terminal Spy or with C-terminal SpyC (Figure 1b-d) and exchanged them into SMM prior to filament assembly.…”

“…Interestingly, this severely interfered with SMM filament assembly in vitro. As proper filaments are required for our experiments, we switched to the SpyTag002 (Spy) SpyCatcher002 (SpyC) system [Keeble et al, 2017] to attach a quantum dot (QD) to the RLC after filaments had already been formed. The 14 amino acid peptide, Spy, spontaneously forms an isopeptide bond with SpyC (15.5 kDa IgG domain), covalently linking the two and anything attached to them.…”

“…Orange boxes represents the RLC sequence (UniProtKB, P02612). Blue boxes represent the SpyTag002 sequence (VPTIVMVDAYKRYK,[Keeble et al, 2017]), black line represents GSGESG linker, and the yellow box represents the SpyCatcher002 sequence(Keeble et al, 2017). Lengths of boxes reflect approximate relative sequence lengths.…”