Power to the protein: enhancing and combining activities using the Spy toolbox

A peptide with simple and selective reactivity expands the function of proteins, from single molecule analysis to potential clinical application.


Spontaneous amidation: kinetic and thermodynamic features
SpyTag/SpyCatcher is a protein coupling approach created by splitting the CnaB2 domain from the bronectin binding protein FbaB from Streptococcus pyogenes. 1 CnaB2 spontaneously forms an intramolecular isopeptide bond between Lys31 and Asp117 (Fig. 1A). SpyCatcher is a 113-residue protein and contains the reactive Lys31. The second part, dubbed SpyTag, is a 13-residue peptide that contains the reactive Asp117 (Fig. 1A). Upon mixing, SpyTag and SpyCatcher associate and spontaneously carry out an amidation reaction promoted by the Spy-Catcher residue Glu77, to form an intermolecular isopeptide bond ( Fig. 1A and B). Spontaneous amidation between SpyTag/ SpyCatcher occurs in a wide-range of temperatures (4-37 C), buffers and pH values. 1 SpyTag and SpyCatcher can be genetically fused to the N-or C-terminus of proteins, and in some cases within internal loops of proteins. 2 Neither moiety contains any cysteine and so it is simple to use in different cellular locations. The reaction is irreversible and proceeds to >99% conversion. 1,3 This approach allows specic covalent coupling of proteins both in vitro and in cells from various species. 2 1.2 Innite affinity: concept, engineering and potential Assembling macromolecular complexes using non-covalent interactions has limited kinetic stability due to the nite value of their dissociation rate-constants. Some covalent interactions, such as disulde bonds, can rapidly rearrange. However, as far as we have been able to measure, the isopeptide bond between SpyTag and SpyCatcher is irreversible. 1 However, such stable reaction has limited utility if the reaction occurs slowly and only in the presence of high concentrations of each partner. Therefore, it is essential to consider each rate-constant. SpyTag and SpyCatcher form an initial non-covalent complex (with association rate-constant k on and dissociation rate-constant k off ), before reacting with a rate-constant k 2 . The best that could happen is a situation termed innite affinity, where k on is diffusion-limited and k 2 is much greater than k off .
The diffusion limit for association of a typical protein:protein complex is 10 5 -10 6 M À1 s À1 . 4 Thus, an innite affinity binding reagent should associate with a k on of 10 5 -10 6 M À1 s À1 and react with a second order rate-constant of 10 5 -10 6 M À1 s À1 .
Through phage display selection and rational design, the SpyTag003/SpyCatcher003 pair now has kinetics approaching innite affinity. SpyTag003/SpyCatcher003 react with a rate of 5.5 Â 10 5 M À1 s À1 , about 400-fold faster than SpyTag/ SpyCatcher (1.4 Â 10 3 M À1 s À1 ) (Fig. 1C). 5,6 Even at low protein concentrations (10 nM), SpyTag003/SpyCatcher003 react close to completion in 15 min, conditions under which little of the original SpyTag/SpyCatcher reacts (Fig. 1C). 6 This rapid reaction opens new potential applications, including for intracellular coupling of poorly expressing proteins on biologically relevant time-scales and enhanced western blot detection. 6

Spy&Go: affinity purication by non-reactive SpyCatcher
An ideal purication tag enables simple and efficient isolation of a protein construct from complex mixtures, but should also enhance the downstream function of that construct. To avoid limitations of the His 6 -tag, such as the toxicity of Ni 2+ , we established the use of SpyTag for protein purication. Spy&Go employs a non-reactive SpyCatcher mutant (SpyDock) to enable purication of proteins containing reactive SpyTags (original, SpyTag002 or SpyTag003) (Fig. 1D). 7 Cell lysate or supernatant containing the SpyTag-linked protein of interest is mixed with SpyDock resin, impurities washed away, and the SpyTag-fusion eluted using high concentrations of imidazole. Spy&Go was shown for purication of proteins from bacterial or mammalian expression. Purication of a dual tagged (His 6 -tag/SpyTag) maltose binding protein by Spy&Go gave a higher purity (98.9 AE 0.5%) than via Ni-NTA purication (66.4 AE 1.9%). 7 Proteins with either N-or C-terminally fused SpyTag as well as with SpyTag inserted in an internal loop could be puried from lysates, with binding capacities of 4-13 mg protein per mL of resin. SpyDock resin was able to be regenerated multiple times and could be stored in 20% ethanol. 7 One of Spy&Go's key initial applications has been the purication of malaria antigens, which can then been be coupled to VLPs as vaccine candidates without an anti-His-tag immune response. 7

Anchoring to surfaces or particles
Classical approaches for anchoring proteins to a surface are hydrophobic adsorption or reaction with one of the many surface amines (from the N-terminus or Lys side-chains). 8 Such approaches lack precision in orientation and oen impair protein function. 9 Alternatively, proteins with a natural or arti-cially introduced surface Cys can be coupled to maleimide or iodoacetyl groups. 8 However, thiol-mediated coupling faces challenges from: (i) competition between coupling and disul-de bond formation, (ii) free Cys interfering with protein secretion, or (iii) promoting misfolding in proteins already containing disulde bonds. 9 For attachment to magnetic beads, the advantage of oriented SpyTag-mediated anchoring of single-domain antibodies was shown, when compared to non-specic attachment by chemical activation of carboxylic acids ( Fig. 2A). 10 SpyTag-mediated anchoring has been applied for protein functionalisation of a range of surfaces, including gold nanoparticles 11 and quantum dots 12 ( Fig. 2A). Anchoring onto a single layer of graphene using SpyTag has been developed for enhancing cryoelectron microscopy structure determination at atomic resolution. 13 SpyTag-linked proteins may be anchored to silica particles assembled through biomimetic silicication 14 or to plastic particles (polyhydroxyalkanoate, PHA) synthesised inside the cell 15 ( Fig. 2A).
To target to the air/water interface, Lynne Regan's group have shown how hydrophobins can still form a monolayer at this interface when linked to SpyTag. 16 Hydrophobin-SpyTag can also assemble proteins of interest around oil droplets, where the particles remained monodisperse for weeks at room temperature. 17 Air bubbles can be nucleated inside cells with genetically-encoded acoustic nanocapsules, which can be decorated using SpyTag/SpyCatcher and provide probes for targeted ultrasound. 18 To address the imprecision of amine-mediated attachment, proteins have been genetically fused to the AviTag peptide and site-specically biotinylated using BirA. 19,20 Biotinylated proteins can then be coupled to streptavidin-linked surfaces, since streptavidin:biotin is one of the strongest non-covalent interactions. However, the streptavidin/biotin linkage is not irreversible, 21 BirA reaction may not reach completion, and biotinylating a protein adds two steps to any pipeline: (i) performing the biotinylation reaction, and (ii) removing free biotin to avoid competition for streptavidin binding sites. Reversibility of streptavidin:biotin is an acute problem when force is applied to the interaction (e.g. $60 pN for > 1 minute when studying protein unfolding), 22 at elevated temperatures, or for long-term storage. Researchers studying the force-dependence of protein interactions and protein folding have sought an interaction that was resistant to force and inextensible. The isopeptide bond runs between the Lys near the N-terminus of SpyCatcher to the Asp in the C-terminus of SpyTag, such that the force passes through the isopeptide bond and does not unfold the rest of the SpyCatcher domain. 23,24 Therefore, SpyTag/SpyCatcher has become a common tool for force spectroscopy, 1 complementing HaloTag's covalent interaction with alkyl halide ligands 22 (Fig. 2B). Proteins for stretching can be fused to SpyTag at any accessible site and SpyCatcher is typically anchored to the solidphase through an N-terminal Cys (Fig. 2B). Such SpyTaganchored stretching has been applied, for example, for testing the mechanical basis of human hearing 25 or for studying the folding pathway of computationally-designed membrane proteins. 26 Force may be measured aer SpyTag anchoring using atomic force microscopy (AFM), optical tweezers, or magnetic tweezers. 27

Control of protein multimerisation state
Natural proteins take on a huge range of multimerisation architectures, 28 from the familiar dimers and tetramers, up to icosahedral architectures with 60 or 180 copies. This multimerisation can have major effects on protein behaviour. Ligand dimerisation is a common way to activate cell signalling. 29 Ligand tetramerisation provides avidity, revealing natural low affinity interactions, e.g. MHC multimers identify antigen-specic T cell populations in infection or cancer. 30 Changing the multimerisation state of a protein of interest has oen depended upon painstaking genetic fusion or tetramerisation of a biotinylated variant using streptavidin. 31 Modular covalent assembly brings the potential to generate one protein of interest bearing SpyTag and then immediately access a toolbox of other protein scaffolds with dened architectures (Fig. 2C).
Low valency assemblies with dihedral symmetry can be accessed through "SpyAvidins" (Fig. 2C). We showed that streptavidin subunits could be fused with SpyCatcher and chimaeric tetramers generated with precisely 1, 2, 3 or 4 SpyCatcher copies. 31 Thereby, ligands could be clustered, as well as interfaced with biotinylated ligands. To access other low valencies using cyclic symmetry, we prepared a set of coiled coil architectures, 7 harnessing structures known in nature or computationally designed by Dek Woolfson's group 32 (Fig. 2C). We applied this coiled coil set, from dimer up to heptamer, to follow valency-dependence of Death Receptor 5 activation of apoptosis. 7 To access higher valencies, we and others have used icosahedral protein architectures (Fig. 2C). SpyCatcher fused to Dodecin from Mycobacterium tuberculosis is a stable 12-mer, which can be quantitatively coupled to SpyTag-fused ligands. 33 SpyCatcher-linked Ferritin has 24 subunits and was used for multimerising tumour neoantigens. 34 SpyCatcher-mi3 is a modied version of computationally-designed 60-mer, which expresses efficiently in Escherichia coli and allowed simple coupling with blood-stage and transmission-blocking malaria antigens. 35 SpyCatcher-AP205, based on a phage capsid, has 180 subunits and can be coupled to antigens related to HIV, tuberculosis and cancer. 36,37 Clustering on viral-like particles leads to a major increase in immunogenicity and may enhance vaccine development for a range of diseases 9 (Fig. 2C). Such SpyTag-based clustering has also been applied to bacterial microcompartments 38 or live viruses, e.g. oncolytic Herpes Simplex Virus, 39 the lamentous viruses Potato Virus X 40 and Tobacco Mosaic Virus, 41 lentivirus 42 or Adeno-associated Virus (AAV). 43,44 Here there is particular interest to change the viral tropism, including retargeting the virus to tumours.
For multimers that can potentially extend innitely, the rst approach using SpyTag was 1D bres made from the bacterial amyloid-forming protein CsgA. 45,46 CsgA-SpyTag was secreted by E. coli to form a living material and generate nanobres decorated with gold nanoparticles or quantum dots. 46 The bres form amyloid networks with extreme stability: the Joshi lab showed how CsgA-SpyCatcher could be isolated from E. coli as a nanoporous mat, survive washing with organic solvent and sodium dodecyl sulfate (SDS), and subsequently still react with SpyTag-fusions. 47 2D surfaces functionalisable via SpyTag (either in vitro or covering living cells) came from fusion to Slayer proteins. 48 3D structures applying SpyTag/SpyCatcher were rst assembled by the groups of David Tirrell and Frances Arnold: hydrogels form upon mixing one protein bearing multiple SpyTags with another protein bearing multiple Spy-Catchers. 49 This gelation enabled stem cell encapsulation; in contrast to other chemistries for hydrogel formation, each component can be precisely functionalised (e.g. with integrin binding sites or matrix metalloproteinase cleavage sites) and the bio-orthogonal reaction leads to high viability of different cell-types. 49 3D networks can also be assembled aer chemical coupling of SpyTag to polymers, for polyethylene glycol (PEG) networks joined by light-induced radical formation. 50

Multiplexing protein function
Proteins with different functionality can oen be connected by genetic fusion. However, challenges arise from: -Increase in folding complexity.
-Mismatch in multimerisation state of each unit.
-Requirement for different post-translational or synthetic chemical modication of each moiety. 9 Therefore there are many situations where modular coupling is preferable. This is especially urgent given the revolutions in Omics and Personalised Medicine, where people are looking for scalable approaches to be applied on 1000 to 100 000 targets. For example, the Human Proteome Project has long had a goal of specic binding reagents for every protein in the human proteome. 51 For each of $20 000 reagents, one would desire a version linked to e.g. 8 different uorophores as well as a probe for ELISA and in vivo imaging (Fig. 3A). With genetic fusion, one requires 20 000 Â 10 constructs to be cloned, expressed and puried, which is impractical even for the largest company. With modular coupling, one requires 20 000 + 10 constructs (Fig. 3A). This concept was put into practice with a cell-free in vitro transcription and translation mix, where binding reagents linked to SpyTag were multiplexed with SpyCatcher-linked uorescent proteins, reporter enzymes or toxins (Fig. 3A). 52 Ron Geyer's lab has developed this concept for multiplexing of antibody formats. 53 Modular assembly with SpyTag/SpyCatcher has also been used to make heterodimeric binders ligating either different cell-surface receptors 54 or different regions of the same receptor. 55 Multicomponent assembly can be enhanced through combining SpyTag/SpyCatcher with the orthogonal SnoopTag/ SnoopCatcher pair, as we applied for 9 ligation steps on a Sepharose solid-phase. The resultant affibody/nanobody teams were tested for synergy in cancer cell killing. 3 CsgA amyloid can also be used as a solid-phase, for sequential assembly of a multi-enzyme pathway for chitin degradation (Fig. 3B). 56 Our group extended 3D network assembly by chemical coupling of SpyTag to the polysaccharide hyaluronic acid, allowing gelation by a protein containing two Spy-Catchers. 57 Using SnoopTag, these hydrogels were independently functionalised with adhesion proteins to modulate behaviour of tumour spheroids. 57 Combining Spy and Snoop pairs also allowed layer-by-layer nanoassembly to harvest uranium from seawater. 58,59 To address the environmental impact of conventional lightemitting diode (LED) phosphors, a white LED was assembled through ligation of uorescent proteins emitting in different parts of the spectrum (Fig. 3C). 60 Similarly, to increase solar energy conversion, plant light harvesting complexes have recently been covalently combined with reaction centres from a purple photosynthetic bacterium to give complementary light absorption. 61

Enzyme resilience and assembly
Enzymes oen require a dynamic structure to bind and release their substrates, as well as for responding to regulatory inputs. Therefore, enzyme stability can be limiting in many situations. For example, enzymes used to enhance digestibility of animal feed must survive treatment with high-pressure steam. 62 Since termini are oen the most exible part of a protein, we tested how locking the termini together through SpyTag/SpyCatcher (to give SpyRings, Fig. 4A) could change the resilience of an enzyme. We were surprised to nd that the effect was oen dramatic, with an enzyme such as b-lactamase retaining nearly all of its solubility and catalytic activity following boiling, if cyclised in this way (Fig. 4A). 63 Similar effects on resilience were found for SpyRing versions of phytase, 64 xylanase 65 and luciferase. 66 Apart from temperature, increased tolerance to organic solvents and denaturant has been found upon SpyRing cyclisation. 67 Another approach to enhance stability and performance has been to encapsulate enzymes in protein cages. Pamela Silver's lab fused two enzymes in indigo biosynthesis via SpyTag/ SpyCatcher on the inside of MS2 phage nanoparticles. This encapsulation enhanced indigo production inside cells, as well as increasing enzyme stability aer 1 week from 95% compared to 5% for free enzymes. 68 Wen-Bin Zhang's lab used p53's dimerisation domain and SpyTag/SpyCatcher to create protein catenanes, generating a dihydrofolate reductase with increased thermal and proteolytic stability. 69 Hydrogels have also been assembled using SpyTag/ SpyCatcher with the enzymes themselves as the construction material. These enzyme networks showed efficient catalytic conversion and good stability for continuous ow biocatalysis (Fig. 4B). 70,71 Bringing together 3 enzymes into a hydrogel with SpyTag/SpyCatcher linked to elastin-like polypeptides increased the yield in Vitamin K2 biosynthesis. 72

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To improve enzyme performance, SpyTag/SpyCatcher may facilitate substrate recruitment. Sortase recognises an -LPXTG motif on a substrate protein and directs ligation to oligoglycine-bearing probes (Fig. 4C). Sortase is oen used at concentrations similar to that of its substrates and efficiency can be limited by sortase's low affinity for -LPXTG. Using -LPXTG linked to SpyTag and Sortase linked to SpyCatcher, the Tsourkas lab enhanced this substrate docking, improving reaction speed and yield (Fig. 4C). 73 Precise positioning and long-term assembly are also important for next-generation DNA sequencing, where SpyTag positions DNA polymerase adjacent to a nanopore and current change reads out nucleic acid sequence (Fig. 4D). 74,75

Cellular applications
The rst paper on SpyTag showed how the peptide could be fused to a cell-surface protein of interest (ICAM-1) for specic labelling on live cells, using SpyCatcher linked to a uorescent dye (Fig. 5A). 1 Subsequent major advances were imaging of channelrhodopsins inside living Caenorhabditis elegans 76 and super-resolution uorescent microscopy inside cells. 77 For imaging inside Saccharomyces cerevisiae, direct fusion to GFP can be problematic for various plasma membrane proteins but labelling via SpyTag/SpyCatcher improved localisation. 78 Fusing SpyTag to a voltage-sensitive dye was applied for uorescent imaging of neuronal action potentials. 79 Various elegant studies used SpyTag to establish membrane translocation in bacterial inner and outer membranes (Fig. 5A). 80,81 A revolution in cancer treatment in recent years has been the clinical success of immunotherapy, using either checkpoint inhibitors or Chimeric Antigen Receptor (CAR)-T cells. 82 CAR-T cells are currently generated by lentiviral transduction, enabling redirection of T cell killing to a cancer-specic target. Modular redirection of CAR-T cells using non-covalent coiled coil assembly at the cell-surface was proposed for tunable T cell activation (e.g. reducing activation in the case of life-threatening cytokine storm) or redirecting T cells to new targets (in the case of immune evasion). 83 Antibody linked to SpyTag has been injected into mice to direct SpyCatcher-expressing CAR-T cells towards ovarian cancer killing (Fig. 5B). Compared to coiled coils, this approach has the potential advantages of the higher stability of the interaction and easier analysis of CAR-T cell conjugation (since coupling survives boiling in SDS). 84 To enhance the study of cell-cell interactions in the immune system, stable transfectants were generated bearing surfaceexposed SpyTag held at various distances from the plasma membrane (Fig. 5C). 85 Target cells expressed a surface protein bearing a twin Strep-tag. Then, questions about cell-cell interactions in terms of ligand length, valency and affinity could be addressed in a modular fashion (without making a huge range of stably transfected cell clones), through titrating in a bridging molecule of SpyCatcher linked to Strep-Tactin (Fig. 5C). 85 Membrane decoration may also be applied for vaccine assembly: Outer Membrane Vesicles (OMVs) from attenuated Salmonella displaying a SpyTag-linked E. coli autotransporter facilitate display of SpyCatcher-linked immunogens (Fig. 5D). 86 In terms of multicellular organisms, SpyTag has been used in silkworms, 87 C. elegans 76 and recently a transgenic mouse. Samantha Harris' lab generated a mouse line with the muscle mechanosensor c-MyBP-C bearing a SpyTag and a tobacco etch virus (TEV) protease cleavage site (Fig. 5E). On detergentpermeabilised muscle cells, her group was able to cut the protein with TEV protease and paste in new SpyCatcher-linked N-terminal regions of the protein. 88 This molecular surgery restored calcium-dependent synchronisation of muscle contraction, but depended on the phosphorylation state of the fragment. We have also used Spy technology to investigate mechanosensing in the cytosol, studying talin's activity at the interface from the extracellular matrix to the cytoskeleton. The cellular function of split talin could be reconstituted by SpyTag003/SpyCatcher003 reaction. 6 However, non-covalent variants of SpyTag003 also allowed sufficient mechanical stability for restoration of cell shape and migration speed. Generating a panel of SpyTag003 variants with decreasing affinity for SpyCatcher003, we could then identify the point at which interaction became insufficient. 6 Spy technology has been applied for various genetic and epigenetic applications. SpyCLIP was developed to map RNAprotein interaction in cells, taking advantage of the irreversible SpyTag interaction to reduce background and improve pulldown efficiency (Fig. 5F). 89 Inside the nucleus, SpyTag has been fused to transcription factors for programmable changing of nucleosome positioning (Fig. 5G) 90 or for a down-sized Cas9 for CRISPR-mediated gene editing. 91

Overview
The dream of synthetic biology for redirecting biological units as reliably as components in an electronic circuit board is still a work in progress. 92 Having simple and reliable ways to bridge proteins to each other or to non-protein components is an important part of achieving that goal. Here we have seen how the community has found many ways to employ SpyTag technology for such challenges. SpyTag may now contribute at each stage in the life of a protein: purication, analysis, and application on cells or ex vivo.

Limitations of SpyTag technology
Despite the range of uses above, SpyTag/SpyCatcher has various limitations: (i) Coupling leaves a molecular scar: the nal construct contains the $17 kDa SpyTag/SpyCatcher. SnoopLigase forms an isopeptide bond between two peptides, giving a smaller molecular scar (the covalently linked SnoopTagJr:DogTag), but requires higher concentration of reactants. 93 Diverse other ligation technologies are available with the advantage of a small scar (e.g. sortase, unnatural amino acid) or no scar (split intein), although facing their own challenges in terms of complexity or limitation to a single terminus. 94 (ii) Reactivity is unregulated: as soon as SpyTag and Spy-Catcher collide, they can react.
(iii) Fusion tolerance: >500 SpyTag/SpyCatcher fusions have been validated (listed in the SpyBank database at https://www2.bioch.ox.ac.uk/howarth/info.htm) and we have published general guidance on the design of linkers and helpful positive and negative controls. 2 Nevertheless, it will help to gain further experience across more compartments and organisms for when fusion to SpyTag or SpyCatcher variants may affect expression yield or perturb natural protein function.
(iv) The more established HaloTag or (strept)avidin:biotin technologies currently have greater infrastructure of commercially-available reagents.
(v) SpyTag/SpyCatcher is non-human and will induce an immune response. 36,95 It is preferable to use a non-human platform for vaccines (to avoid autoimmunity). However, immunogenicity will be a challenge to SpyTag's use in therapeutics.

Future directions
Although there have been a range of interesting cellular studies, the majority of applications of SpyTag has been in vitro. The moderate reaction rate of SpyTag/SpyCatcher has limited the time resolution and labelling efficiency for proteins at low concentration. The 400-fold accelerated reaction of SpyTag003/ SpyCatcher003 (ref. 6) may facilitate many more applications of covalent decoration in cells and organisms.
We recently established a panel of non-covalent SpyTag/ SpyCatcher complexes with affinities spanning 20 nM to >1 mM, to test the stability requirements of a force-dependent cytosolic interaction. 6 Being able to compare how cells respond when an interaction is irreversible, slowly dynamic or rapidly dynamic may be useful in various other biological contexts. Promising vaccine applications of Spy technology have been shown in animal models, 9 so it will be important to evaluate how these platforms perform in the clinic. With the combination of recent advances in the underlying technology and a widening scope of applications, it will be exciting to see how the Spy toolbox develops in the future.