Engineered Binding Protein Research Articles

Evaluation of the Effect of β-Wrapin AS69 in a Mouse Model Based on Alpha-Synuclein Overexpression.

Aggregation of the protein α-Synuclein (αSyn) is a hallmark of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy, and alleviating the extent of αSyn pathology is an attractive strategy against neurodegeneration. The engineered binding protein β-wrapin AS69 binds monomeric αSyn. AS69 reduces primary and secondary nucleation as well as fibril elongation in vitro. It also mitigates aSyn pathology in a mouse model based on intrastriatal injection of aSyn pre-formed fibrils (PFFs). Since the PFF-based model does not represent all aspects of PD, we tested here whether AS69 can reduce neurodegeneration resulting from αSyn overexpression. Human A53T-αSyn was overexpressed in the mouse Substantia nigra (SN) by using recombinant adeno-associated viral vector (rAAV). AS69 was also expressed by rAAV transduction. Behavioral tests and immunofluorescence staining were used as outcomes. Transduction with rAAV-αSyn resulted in αSyn pathology as reported by phospho-αSyn staining and caused degeneration of dopaminergic neurons in the SN. The co-expression of rAAV-AS69 did not reduce αSyn pathology or the degeneration of dopaminergic neurons. We conclude that αSyn monomer binding by rAAV-AS69 was insufficient to protect from aSyn pathology resulting from αSyn overexpression.

Affimer reagents as tool molecules to modulate platelet GPVI-ligand interactions and specifically bind GPVI dimer

AbstractGlycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Sequence-based identification of amyloidogenic β-hairpins reveals a prostatic acid phosphatase fragment promoting semen amyloid formation

β-Structure-rich amyloid fibrils are hallmarks of several diseases, including Alzheimer’s (AD), Parkinson’s (PD), and type 2 diabetes (T2D). While amyloid fibrils typically consist of parallel β-sheets, the anti-parallel β-hairpin is a structural motif accessible to amyloidogenic proteins in their monomeric and oligomeric states. Here, to investigate implications of β-hairpins in amyloid formation, potential β-hairpin-forming amyloidogenic segments in the human proteome were predicted based on sequence similarity with β-hairpins previously observed in Aβ, α-synuclein, and islet amyloid polypeptide, amyloidogenic proteins associated with AD, PD, and T2D, respectively. These three β-hairpins, established upon binding to the engineered binding protein β-wrapin AS10, are characterized by proximity of two sequence segments rich in hydrophobic and aromatic amino acids, with high β-aggregation scores according to the TANGO algorithm. Using these criteria, 2505 potential β-hairpin-forming amyloidogenic segments in 2098 human proteins were identified. Characterization of a test set of eight protein segments showed that seven assembled into Thioflavin T-positive aggregates and four formed β-hairpins in complex with AS10 according to NMR. One of those is a segment of prostatic acid phosphatase (PAP) comprising amino acids 185–208. PAP is naturally cleaved into fragments, including PAP(248−286) which forms functional amyloid in semen. We find that PAP(185−208) strongly decreases the protein concentrations required for fibril formation of PAP(248−286) and of another semen amyloid peptide, SEM1(86−107), indicating that it promotes nucleation of semen amyloids. In conclusion, β-hairpin-forming amyloidogenic protein segments could be identified in the human proteome with potential roles in functional or disease-related amyloid formation.

Computational design of a β-wrapin's N-terminal domain with canonical and non-canonical amino acid modifications mimicking curcumin's proposed inhibitory function

β-wrapins are engineered binding proteins of which different mutants can bind and sequester amyloidogenic proteins amyloid-β (Aβ), islet amyloid polypeptide (IAPP), and α-synuclein (α-syn), thereby inhibiting their aggregation into amyloid fibrils. β-wrapin AS10 is capable of binding and sequestering all three amyloidogenic monomers with micro-molar affinity, with its N-terminal domains remaining flexible and non-functional. Here, we computationally investigated the hypothesis that the anti-amyloid properties of AS10 can be amplified by redesigning its currently non-functional N-terminal domain with particular combinations of canonical and non-canonical amino acids (ncAAs) that can mimic the binding and inhibitory anti-amyloid function of curcumin, using a combination of molecular docking and molecular dynamics simulations. Our simulations suggest that the inhibitory mechanism attributed to the binding of the computationally designed AS10 N-terminal domain to the Aβ fibril can act simultaneously to its sequestering properties for Aβ which are attributed to the core of AS10. Thus, our study proposes that the N-terminal domain of AS10 can be further modified to amplify its anti-amyloid properties, resulting in a β-wrapin that may simultaneously prohibit elongation to existing amyloid fibrils and also sequester amyloid monomers.

Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein–protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal β-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

Functional Comparison of Bioactive Cellulose Materials Incorporating Engineered Binding Proteins.

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers. Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

β-Turn exchanges in the α-synuclein segment 44-TKEG-47 reveal high sequence fidelity requirements of amyloid fibril elongation

The folding of turns and β-hairpins has been implicated in amyloid formation, with diverse potential consequences such as promotion or inhibition of fibril nucleation, fibril elongation, or off-pathway oligomer formation. In the Parkinson's disease-associated protein α-synuclein (αS), a β-hairpin comprised of residues 36–56 was detected in complex with an engineered binding protein, with a turn formed by the αS sequence segment 44-TKEG-47. Molecular dynamics simulations revealed extensive populations of transient β-hairpin conformations in this region in free, monomeric αS. Here, we investigated potential effects of turn formation on αS fibril formation by studying the aggregation kinetics of an extensive set of αS variants with between two and four amino acid exchanges in the 44-TKEG-47 segment. The exchanges were chosen to specifically promote formation of β1-, β1’-, or β2’-turns. All variants assembled into amyloid fibrils, with increased β1’- or β2’-turn propensity associated with faster aggregation and increased β1-turn propensity with slower aggregation compared to wild-type (WT) αS. Atomic force microscopy demonstrated that β-turn exchanges altered fibril morphology. In cross-elongation experiments, the turn variants showed a low ability to elongate WT fibril seeds, and, vice versa, WT monomer did not efficiently elongate turn variant fibril seeds. This demonstrates that sequence identity in the turn region is crucial for efficient αS fibril elongation. Elongation experiments of WT fibril seeds in the presence of both WT and turn variant monomers suggest that the turn variants can bind and block WT fibril ends to different degrees, but cannot efficiently convert into the WT fibril structure. Our results indicate that modifications in the 44-TKEG-47 segment strongly affect amyloid assembly by driving αS into alternative fibril morphologies, whose elongation requires high sequence fidelity.

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

Highly sensitive immunodiagnostics at the point of care employing alternative recognition elements and smartphones: hype, trend, or revolution?

Immunodiagnostic tests performed at the point of care (POC) today usually employ antibodies for biorecognition and are read out either visually or with specialized equipment. Availability of alternative biorecognition elements with promising features as well as smartphone-based approaches for signal readout, however, challenge the described established configuration in terms of analytical performance and practicability. Assessing these developments' clinical relevance and their impact on POC immunodiagnostics is demanding. The first part of this review will therefore give an overview on suitable diagnostic biosensors based on alternative recognition elements (such as nucleic acid-based aptamers or engineered binding proteins) and exemplify advantages and drawbacks of these biomolecules on the base of selected assays. The second part of the review then focuses on smartphone-connected diagnostics and discusses the indispensable considerations required for successful future clinical POCT implementation. Together, the joint depiction of two of the most innovative and exciting developments in the field will enable the reader to cast a glance into the distant future of POC immunodiagnostics.

Targeted Protein Degradation through Cytosolic Delivery of Monobody Binders Using Bacterial Toxins.

Monobodies are small engineered binding proteins that, upon expression in cells, can inhibit signaling of cytosolic oncoproteins with outstanding selectivity. Efficacy may be further increased by inducing degradation of monobody targets through fusion to the von Hippel–Lindau (VHL) substrate receptor of the Cullin2-E3 ubiquitin ligase complex. However, potential therapeutic use is currently limited, because of the inability of monobody proteins to cross cellular membranes. Here, we use a chimeric bacterial toxin, composed of the Shiga-like toxin B (Stx2B) subunit and the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA-II) for delivery of VHL–monobody protein fusions to target endogenous tyrosine kinases in cancer cells. Depending on the expression of the Stx2B receptor Gb3 on the cell surface, we show that monobodies are taken up by an endocytic route, but are not degraded in lysosomes. Delivery of monobodies fused to a nuclear localization signal resulted in accumulation in the nucleus, thereby indirectly, but unequivocally, demonstrating cytosolic delivery. Delivery of VHL fused to monobodies targeting the Lck tyrosine kinase in T-cells resulted in reduced Lck protein levels, which was dependent on the expression of Gb3. This led to the inhibition of proximal signaling events downstream of the T-cell receptor complex. This work provides a prime example of the delivery of a stoichiometric protein inhibitor of an endogenous target protein to cells and inducing its degradation without the need of genetic manipulation of target cells. It lays the foundation for further in vivo exploitation of this delivery system.

Elucidating the multi-targeted anti-amyloid activity and enhanced islet amyloid polypeptide binding of β-wrapins

β-wrapins are engineered binding proteins stabilizing the β-hairpin conformations of amyloidogenic proteins islet amyloid polypeptide (IAPP), amyloid-β, and α-synuclein, thus inhibiting their amyloid propensity. Here, we use computational and experimental methods to investigate the molecular recognition of IAPP by β-wrapins. We show that the multi-targeted, IAPP, amyloid-β, and α-synuclein, binding properties of β-wrapins originate mainly from optimized interactions between β-wrapin residues and sets of residues in the three amyloidogenic proteins with similar physicochemical properties. Our results suggest that IAPP is a comparatively promiscuous β-wrapin target, probably due to the low number of charged residues in the IAPP β-hairpin motif. The sub-micromolar affinity of β-wrapin HI18, specifically selected against IAPP, is achieved in part by salt-bridge formation between HI18 residue Glu10 and the IAPP N-terminal residue Lys1, both located in the flexible N-termini of the interacting proteins. Our findings provide insights towards developing novel protein-based single- or multi-targeted therapeutics.

High-throughput characterization of protein–protein interactions by reprogramming yeast mating

High-throughput methods for screening protein-protein interactions enable the rapid characterization of engineered binding proteins and interaction networks. While existing approaches are powerful, none allow quantitative library-on-library characterization of protein interactions in a modifiable extracellular environment. Here, we show that sexual agglutination of Saccharomyces cerevisiae can be reprogrammed to link interaction strength with mating efficiency using synthetic agglutination (SynAg). Validation of SynAg with 89 previously characterized interactions shows a log-linear relationship between mating efficiency and protein binding strength for interactions with Kds ranging from below 500 pM to above 300 μM. Using induced chromosomal translocation to pair barcodes representing binding proteins, thousands of distinct interactions can be screened in a single pot. We demonstrate the ability to characterize protein interaction networks in a modifiable environment by introducing a soluble peptide that selectively disrupts a subset of interactions in a representative network by up to 800-fold. SynAg enables the high-throughput, quantitative characterization of protein-protein interaction networks in a fully defined extracellular environment at a library-on-library scale.

Structure of human islet amyloid polypeptide in complex with an engineered binding protein

Structure of human islet amyloid polypeptide in complex with an engineered binding protein

β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor.

In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation.

Activity-based assessment of an engineered hyperthermophilic protein as a capture agent in paper-based diagnostic tests.

Antibodies have traditionally served as the affinity reagents of choice in point-of-care diagnostic biosensors. However, this class of proteins is not ideally suited for this use, being poorly characterized and prone to thermal denaturation. Here, we present an activity-based assessment of an alternative engineered binding protein in a cellulose-based assay.

Impact of subunit linkages in an engineered homodimeric binding protein to α-synuclein.

Aggregation of the protein α-synuclein (α-syn) has been implicated in Parkinson's disease and other neurodegenerative disorders, collectively referred to as synucleinopathies. The β-wrapin AS69 is a small engineered binding protein to α-syn that stabilizes a β-hairpin conformation of monomeric α-syn and inhibits α-syn aggregation at substoichiometric concentrations. AS69 is a homodimer whose subunits are linked via a disulfide bridge between their single cysteine residues, Cys-28. Here we show that expression of a functional dimer as a single polypeptide chain is achievable by head-to-tail linkage of AS69 subunits. Choice of a suitable linker is essential for construction of head-to-tail dimers that exhibit undiminished α-syn affinity compared with the solely disulfide-linked dimer. We characterize AS69-GS3, a head-to-tail dimer with a glycine-serine-rich linker, under oxidized and reduced conditions in order to evaluate the impact of the Cys28-disulfide bond on structure, stability and α-syn binding. Formation of the disulfide bond causes compaction of AS69-GS3, increases its thermostability, and is a prerequisite for high-affinity binding to α-syn. Comparison of AS69-GS3 and AS69 demonstrates that head-to-tail linkage promotes α-syn binding by affording accelerated disulfide bond formation.

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337-342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.

The Off-rate of Monomers Dissociating from Amyloid-β Protofibrils

The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.

High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule

Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K(D), of ~20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)2htt dimer binds with an off-rate of k(off) ≤ 5 × 10(-6) s(-1) and an estimated K(D) ≤ 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.