iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Design and Evolution of Enhanced Peptide-Peptide Ligation for Modular Transglutaminase Assembly.

Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments.

Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2.

Adenovirus vector vaccines have been widely and successfully deployed in response to coronavirus disease 2019 (COVID-19). However, despite inducing potent T cell immunity, improvement of vaccine-specific antibody responses upon homologous boosting is modest compared with other technologies. Here, we describe a system enabling modular decoration of adenovirus capsid surfaces with antigens and demonstrate potent induction of humoral immunity against these displayed antigens. Ligand attachment via a covalent bond was achieved using a protein superglue, DogTag/DogCatcher (similar to SpyTag/SpyCatcher), in a rapid and spontaneous reaction requiring only co-incubation of ligand and vector components. DogTag was inserted into surface-exposed loops in the adenovirus hexon protein to allow attachment of DogCatcher-fused ligands on virus particles. Efficient coverage of the capsid surface was achieved using various ligands, with vector infectivity retained in each case. Capsid decoration shielded particles from vector neutralizing antibodies. In prime-boost regimens, adenovirus vectors decorated with the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike induced >10-fold higher SARS-CoV-2 neutralization titers compared with an undecorated vector encoding spike. Importantly, decorated vectors achieved equivalent or superior T cell immunogenicity against encoded antigens compared with undecorated vectors. We propose capsid decoration using protein superglues as a novel strategy to improve efficacy and boostability of adenovirus-based vaccines and therapeutics.

Approaching infinite affinity through engineering of peptide-protein interaction.

Much of life's complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide-protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen-deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide-protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation.

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.

Programmable polyproteams built using twin peptide superglues.

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.

Smart superglue in streptococci? The proof is in the pulling.

Gram-positive bacteria use reactive thioester-containing proteins to form covalent bonds, which may enable strong adhesion to host surfaces, but how these proteins selectively adhere to different surfaces is not clear. The Editors' Pick by Echelman et al. applied single-molecule force spectroscopy to show that an adhesin protein can regenerate its thioester in the absence of pulling. This selective interaction would represent a new principle of mechanical proof-reading, whereby only reactions supporting anchorage of the bacterium are maintained.

SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation.

Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability. After engineering, SnoopLigase demonstrated high-yield coupling under a wide range of buffers and temperatures. SnoopTagJr and DogTag were functional at the N- or C-terminus, while DogTag was also functional at internal sites in proteins. Having directed reaction of SnoopTagJr and DogTag, SnoopLigase remained stably bound to the ligated product, thus reconstituting the parent domain. Separating products from unreacted starting material and catalyst is often as challenging as reactions themselves. However, solid-phase immobilization of SnoopLigase enabled the ligated SnoopTagJr-DogTag product to be eluted with high purity, free from SnoopLigase or unligated substrates. The solid-phase catalyst could then be reused multiple times. In search of a generic route to improve the resilience of enzymes, we fused SnoopTagJr to the N-terminus and DogTag to the C-terminus of model enzymes, allowing cyclization via SnoopLigase. While wild-type phytase and ß-lactamase irreversibly aggregated upon heating, cyclization using SnoopLigase conferred exceptional thermoresilience, with both enzymes retaining solubility and activity following heat treatment up to 100 °C. SnoopLigase should create new opportunities for conjugation and nanoassembly, while illustrating how to harness product inhibition and extend catalyst utility.

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems.

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.

SpyLigase peptide-peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture.

Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics.

SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform 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 a wide range of conditions and at either protein terminus. SpyCatcher002 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.

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

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.

The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:ß2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.

Separating speed and ability to displace roadblocks during DNA translocation by FtsK.

FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.

Love-Hate ligands for high resolution analysis of strain in ultra-stable protein/small molecule interaction.

The pathway of ligand dissociation and how binding sites respond to force are not well understood for any macromolecule. Force effects on biological receptors have been studied through simulation or force spectroscopy, but not by high resolution structural experiments. To investigate this challenge, we took advantage of the extreme stability of the streptavidin-biotin interaction, a paradigm for understanding non-covalent binding as well as a ubiquitous research tool. We synthesized a series of biotin-conjugates having an unchanged strong-binding biotin moiety, along with pincer-like arms designed to clash with the protein surface: 'Love-Hate ligands'. The Love-Hate ligands contained various 2,6-di-ortho aryl groups, installed using Suzuki coupling as the last synthetic step, making the steric repulsion highly modular. We determined binding affinity, as well as solving 1.1-1.6Å resolution crystal structures of streptavidin bound to Love-Hate ligands. Striking distortion of streptavidin's binding contacts was found for these complexes. Hydrogen bonds to biotin's ureido and thiophene rings were preserved for all the ligands, but biotin's valeryl tail was distorted from the classic conformation. Streptavidin's L3/4 loop, normally forming multiple energetically-important hydrogen bonds to biotin, was forced away by clashes with Love-Hate ligands, but Ser45 from L3/4 could adapt to hydrogen-bond to a different part of the ligand. This approach of preparing conflicted ligands represents a direct way to visualize strained biological interactions and test protein plasticity.

SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme.

SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of ß-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized ß-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

SpyMask enables combinatorial assembly of bispecific binders.

Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.