Multivalent Protein Research Articles

Multivalent binding proteins can drive collapse and reswelling of chromatin in confinement.

Collapsed conformations of chromatin have been long suspected of being mediated by interactions with multivalent binding proteins, which can bring together distant sections of the chromatin fiber. In this study, we use Langevin dynamics simulation of a coarse grained chromatin polymer to show that the role of binding proteins can be more nuanced than previously suspected. In particular, for chromatin polymer in confinement, entropic forces can drive reswelling of collapsed chromatin with increasing binder concentrations, and this reswelling transition happens at physiologically relevant binder concentrations. Both the extent of collapse, and also of reswelling depends on the strength of confinement. We also study the kinetics of collapse and reswelling and show that both processes occur in similar timescales. We characterise this reswelling of chromatin in biologically relevant regimes and discuss the non-trivial role of multivalent binding proteins in mediating the spatial organisation of the genome.

Targeted delivery of oligonucleotides using multivalent protein-carbohydrate interactions.

Cell surface protein-carbohydrate interactions are essential for tissue-specific recognition and endocytosis of viruses, some bacteria and their toxins, and many glycoproteins. Often protein-carbohydrate interactions are multivalent - multiple copies of glycans bind simultaneously to multimeric receptors. Multivalency enhances both affinity and binding specificity, and is of interest for targeted delivery of drugs to specific cell types. The first such example of carbohydrate-mediated drug delivery to reach the clinic is Givosiran, a small interfering ribonucleic acid (siRNA) that is conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand. This ligand enables efficient uptake of the nucleic acid by the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Synthetic multivalent ligands for ASGP-R were among the first 'cluster glycosides' developed at the birth of multivalent glycoscience around 40 years ago. In this review we trace the history of 'GalNAc targeting' from early academic studies to current pharmaceuticals and consider what other opportunities could follow the success of this delivery technology.

Rational design of multivalent biosensor surfaces to enhance viral particle capture.

Viral particles bind to receptors through multivalent protein interactions. Such high avidity interactions on sensor surfaces are less studied. In this work, three polyelectrolytes that can form biosensing surfaces with different interfacial characteristics in probe density and spatial arrangement were designed. Quartz crystal microbalance, interferometry and atomic force microscopy were used to study their surface density and binding behaviors with proteins and virus particles. A multivalent adsorption kinetic model was developed to estimate the number of bonds from the viral particles bound to the polyelectrolyte surfaces. Experimental results show that the heterogeneous 3D surface with jagged forest-like structure enhances the virus capture ability by maximizing the multivalent interactions. As a proof of concept, specific coronavirus detection was achieved in spiked swab samples. These results indicate the importance of both probe density and their spatial arrangement on the sensing performance, which could be used as a guideline for rational biosensing surface design.

Multivalent forms of the ribonuclease H1 hybrid binding domain are high-affinity binders of RNA-DNA hybrids.

The hybrid binding domain (HBD) is a conserved fold present in ribonucleases H1 that selectively recognizes RNA-DNA hybrids, which are structures present in cellular R-loops and participate in diverse biological processes. We engineered multivalent HBD proteins to create high-affinity hybrid binders. Using EMSA- and SPR-based analyses, we showed that the triple-HBD protein exhibits a ~ 22 000-fold increase in hybrid affinity (KD 370 pm) relative to the single HBD (KD 8.29 μm), with the length and sequence of the linkers enabling optimal function. These findings provide a framework for testing models that correlate multivalency and affinity to understand how multivalent proteins function and also can serve to guide applications that exploit multivalency as a strategy to enhance binding affinity.

Translational regulation by Hfq–Crc assemblies emerges from polymorphic ribonucleoprotein folding

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post‐transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo‐EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA‐specific quaternary architectures resulting from the combination of multivalent protein–protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.

Multivalent binding kinetics resolved by fluorescence proximity sensing

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

The stoichiometric interaction model for mesoscopic MD simulations of liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) has received considerable attention in recent years for explaining the formation of cellular biomolecular condensates. The fluidity and the complexity of their components make molecular simulation approaches indispensable for gaining structural insights. Domain-resolution mesoscopic model simulations have been explored for cases in which condensates are formed by multivalent proteins with tandem domains. One problem with this approach is that interdomain pairwise interactions cannot regulate the valency of the binding domains. To overcome this problem, we propose a new potential, the stoichiometric interaction (SI) potential. First, we verified that the SI potential maintained the valency of the interacting domains for the test systems. We then examined a well-studied LLPS model system containing tandem repeats of SH3 domains and proline-rich motifs. We found that the SI potential alone cannot reproduce the phase diagram of LLPS quantitatively. We had to combine the SI and a pairwise interaction; the former and the latter represent the specific and nonspecific interactions, respectively. Biomolecular condensates with the mixed SI and pairwise interaction exhibited fluidity, whereas those with the pairwise interaction alone showed no detectable diffusion. We also compared the phase diagrams of the systems containing different numbers of tandem domains with those obtained from the experiments and found quantitative agreement in all but one case.

Development of a thermostable SARS-CoV-2 variant-based bivalent protein vaccine with cross-neutralizing potency against Omicron subvariants

SARS-CoV-2 variants have posed significant challenges to the hopes of using ancestral strain-based vaccines to address the risk of breakthrough infection by variants. We designed and developed a bivalent vaccine based on SARS-CoV-2 Alpha and Beta variants (named SCTV01C). SCTV01C antigens were stable at 25 oC for at least 6 months. In the presence of a squalene-based oil-in-water adjuvant SCT-VA02B, SCTV01C showed significant protection efficacy against antigen-matched Beta variant, with favorable safety profiles in rodents. Notably, SCTV01C exhibited cross-neutralization capacity against Omicron subvariants (BA.1, BA.1.1, BA.2, BA.3, and BA.4/5) in mice, superior to a WT (D614G)-based vaccine, which reinforced our previously published findings that SCTV01C exhibited broad-spectrum neutralizing potencies against over a dozen pre-Omicron variants and the Omicron BA.1 variant. In summary, variant-based multivalent protein vaccine could be a platform approach to address the challenging issues of emerging variants, vaccine hesitancy and the needs of affordable and thermal stable vaccines.

Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library.

The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\\ml and 9.6 ng\\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

Affinity and Valence Impact the Extent and Symmetry of Phase Separation of Multivalent Proteins.

Biomolecular self-assembly spatially segregates proteins with a limited number of binding sites (valence) into condensates that coexist with a dilute phase. We develop a many-body lattice model for a three-component system of proteins with fixed valence in a solvent. We compare the predictions of the model to experimental phase diagrams that we measure invivo, which allows us to vary specifically a binding site's affinity and valency. We find that the extent of phase separation varies exponentially with affinity and increases with valency. Valency alone determines the symmetry of the phase diagram.

A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition.

Fully substituted peptide/[60]fullerene hexakis-adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis-adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis-adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E-selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition

AbstractFully substituted peptide/[60]fullerene hexakis‐adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis‐adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis‐adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E‐selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

Design of Specific Nucleic Acid‐Based Biosensors for Protein Binding Activity

AbstractNucleic acid‐based biosensors for the detection of specific proteins combine the typical programmability of synthetic DNA systems with artificially controlled DNA‐protein communication. The high‐affinity interaction between a target protein and a specific ligand, such as an aptamer sequence, or a double stranded DNA domain, or a small peptide, is paired with a nature‐mimicking molecular mechanism allowing for probing, processing, and translating protein binding activity into a measurable signal. In this Review, two main strategies developed in the context of protein‐responsive nucleic acid‐based biosensors are discussed. One is the design of proximity‐based assays harnessing the spatial colocalization of functional probes within the volume of a multivalent protein. The other is the engineering of dynamic DNA structures that undergo a controlled conformational or structural change upon protein binding. Examples of applications from optical and electrochemical detection of antibodies in biofluids to fluorescence imaging of transcription factors in living cells are reported, and suggestions along with possible future directions in the field are discussed.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Designing ferritin nanocage based vaccine candidates for SARS-CoV-2 by in silico engineering of its HLA I and HLA II epitope peptides

New variants of SARS-CoV-2 are continuously being reported. To curtail the spread of this virus, it is essential to find an efficient and potent vaccine. Here, we report in silico designing of a protein (ferritin: FR) nanocage fused with multiple epitopes identified using the immuno-informatics approach and high-throughput screening. Employing computational approaches, we identified potential epitopes from membrane, nucleocapsid, and envelope proteins of SARS-CoV-2 and docked them on the selected human leukocyte antigen Class I and II receptors, then the stability of the complexes was assessed using molecular dynamics simulation studies. We have engineered chimeric ferritin nanocage, chm66FR, with the nested peptide of 10 epitopes by replacing the loop region at the 66th position of the nanocage, then its stability was confirmed using metadynamics simulation. Further, we used the homotrimeric ‘6-helical bundle’ of the spike protein to engineer the chimeric 6HB (chm6HB). The chm6HB is, engineered with three epitope peptides, mounted on the N-terminal trimeric interface of the chm66FR to generate the chm6HB-chm66FR, which contains 15 epitope peptides. Chimeric FR nanocages and the chm6HB could be potential vaccine candidates against strains of SARS-CoV-2. These multivalent and multiple epitopes protein nanocages and scaffolds could mount both humoral and T-cell mediated immune responses against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Abstract 4157: Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability

Abstract Purpose: There is a critical need in immunotherapy drug development to enable focused and sustained immune cell modulation within a tumor to induce and propagate a system-wide anti-tumor response. We have developed a novel immunotherapy platform that could be used to generate geographically focused cancer cell growth inhibition or immune cell activation, thereby stimulating an anti-tumor immune response against primary solid tumors that can also travel to secondary metastases. Methods: Using published methods, we synthesized multivalent protein (MVP) conjugates by conjugating multiple copies (i.e. valency) of immune stimulating proteins, checkpoint inhibitors or anti-tumor antibodies to soluble, long-chain biopolymers. We verified that we can reproducibly generate MVP valencies ranging from 20-120 protein copies (±10%) per polymer backbone. We determined the binding affinity of these MVPs to their respective targets using biolayer interferometry and cell bioassays, and we measured the hydrodynamic radius of these immunotherapies using dynamic light scattering. Then, we injected fluorescently modified MVPs or their unconjugated counterparts directly into a variety of solid tumor models in mice. By taking longitudinal in vivo fluorescence measurements of the intratumoral (IT) drug signal over multiple days, we measured the IT half-life of each treatment. Results: Based on binding affinity measurements, we found that MVP potency increased directly with protein valency, and at high valency, the potency of MVPs were substantially greater than the unconjugated protein controls. Multivalent conjugation also increased the hydrodynamic radius of the MVPs to at least ten times larger than the unconjugated therapeutics. This large size was sufficient to slow the diffusion of MVP immunotherapies through dense tissues, such as solid tumors, as demonstrated by our in vivo studies. MVPs exhibited a higher IT drug signal with a more durable gradient within the tumor compared to the unconjugated controls, resulting in an extension of their IT half-lives by >5X in mouse solid tumors. Conclusions: The MVP platform can be used to modulate the potency and therapeutic durability for a wide range of immunotherapy targets. Further, the MVPs stay focused within the tumor after IT injection where they could generate a sustained anti-tumor immune response with minimal systemic exposure. Therefore, we expect MVP immunotherapies to have a better safety profile than IT or systemic delivery of an unconjugated therapeutic. We will continue to develop our internal MVP pipeline to finalize a candidate for IND-enabling studies. We are also seeking collaborations for co-development of additional immunotherapies that could benefit from the extended IT exposure and potency modulation enabled by the MVP platform. Citation Format: Livia Brier, Amy A. Twite, Adam Barnebey, Mavish Mahomed, Wesley M. Jackson. Using a multivalent immunotherapy platform to extend intratumoral therapeutic durability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4157.

Multivalent protein–protein interactions are pivotal regulators of eukaryotic Hsp70 complexes

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70’s binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein–protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both “canonical” interactions, which are universally conserved, and “non-canonical” (or “secondary”) contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70’s secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.

Tuning Surface Plasmon Resonance Responses through Size and Crosslinking Control of Multivalent Protein Binding-Capable Nanoscale Hydrogels.

Surface plasmon resonance (SPR) phenomena have been widely studied to detect biomolecules because of their high sensitivity and ability to determine biomolecular interactions with kinetic information. However, highly selective detection in specific concentration ranges relevant to target biomolecules is still a challenging task. Recently, we developed bioresponsive nanoscale hydrogels to selectively intensify SPR signals through multivalent protein binding (MPB) events with target biomolecules, including IL-2, where we were able to demonstrate exceptional selectivity for target biomolecules with minimal responses to nonspecific and monovalent binding events. In this work, we systematically explored the relationship between the physical properties of MPB-capable nanoscale hydrogels and their SPR response induced in the presence of the programmed cell death protein 1 antibody (PD-1Ab) as a model target biomolecule. First, we developed a synthetic protocol by controlling various reaction parameters to construct a library of nanoscale poly(N-isopropylacrylamide-co-acrylic acid) hydrogels (NHs) with different sizes (from 400 nm to 1 μm) and degrees of crosslinking (from 2 to 8%). Then, by incorporating MPB-capable PD-1 receptors onto the surface of NHs to form PD-1-responsive nanoscale hydrogels (PNHs), the hydrogel size and crosslinking dependency of their SPR responses were investigated. Our results reveal the appropriate hydrogel size regime and degree of crosslinking for effective PD-1Ab detection at specific concentrations range between a few nM and 1 μM. Overall, our study demonstrates that by tuning the physical properties of the nanoscale hydrogel matrix, the sensitivity and detection range of MPB-based SPR sensors can be modulated to potentially benefit clinical applications such as monitoring diverse therapeutic biomolecules.

Membrane Cargo Density-Dependent Interaction between Protein and Lipid Domains on the Giant Unilamellar Vesicles.

Protein cargos anchored on the lipid membrane can be segregated by fluidic domain phase separation. Lipid membranes at certain compositions may separate into lipid domains to segregate cargos, and protein cargos themselves may be involved in protein condensate domain formation with multivalent binding proteins to segregate cargos. Recent studies suggest that these two driving forces of phase separation closely interact on the lipid membranes to promote codomain formation. In this report, we studied the effect of cargo density on the outcome of the cargo phase separation on giant unilamellar vesicles. Proteins and lipids are connected only by the anchored cargos, so it was originally hypothesized that higher cargo density would increase the degree of interaction between the lipid and protein domains, promoting more phase separation. However, fluorescence image analysis on different cargo densities showed that the cooperative domain formation and steric pressure are at a tug of war opposing each other. Cooperative domain formation is dominant under lower anchor density conditions, and above a threshold density, steric pressure was dominant opposing the domain formation. The result suggests that the cargo density is a key parameter affecting the outcome of cargo organization on the lipid membranes by phase separation.