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Recycling antibodies can bind to their target antigen at neutral pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the antibodies to be recycled into circulation via FcRn-mediated pathway, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve the same therapeutic effect at lower doses than their non-recyclable counterparts. The development of such antibodies is typically achieved by histidine doping of the variable regions of specific antibodies or by performingin vitroantibody selection campaigns utilizing histidine doped libraries. While often successful, these strategies may introduce sequence liabilities, as they often involve mutations that may render the resultant antibodies to be non-natural. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom ofNaja kaouthiain a pH-dependent manner. Upon screening of the discovered antibodies with immunoassays and bio-layer interferometry, a pH-dependent antibody was discovered that exhibits an 8-fold higher dissociation rate at pH 5.5 than 7.4. Interestingly, the variable domains of the pH-dependent antibody were found to be entirely devoid of histidines, demonstrating that pH-dependency may not always be driven by this amino acid. Further, given the high diversity available in a naïve antibody library, the methodology presented here can likely be applied to discover pH-dependent antibodies against different targetsab initiowithout the need of histidine doping.For broader audienceHere, we present the discovery of an α-cobratoxin targeting pH-dependent antibody, with a variable region devoid of histidines, from a naïve antibody library with natural variable domains. Our findings suggest that the commonly taken approach of histidine doping to find pH-dependent antibodies may not always be required, and thus offer an alternative strategy for the discovery of pH-dependent antibodies.

Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins, and comparisons using proteins with poor reference templates are lacking. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures with no reference templates. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins that have poor reference templates, are large, and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.StatementRecent advances in machine learning have led to the development of new protein structure prediction tools. However, these tools have mainly been tested on well-known proteins and their performance on proteins without known templates is unclear. This study evaluated the performance of three tools on over 1000 snake venom toxins. We found that while caution is required when studying poorly characterised proteins, these tools offer valuable opportunities to understand protein function and applications.

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 cobra Naja 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.

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Throughout history, pandemics like the Black Death or the Spanish Flue have periodically wiped out large parts of populations. Today, history repeats itself with the on-going SARS-CoV-2 pandemic, w...

In vitro antibody selection against pathogens from naive combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection 1–4 . Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction 5 . Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naive human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Die Ehlers-Danlos-Syndrome (EDS) sind eine Gruppe seltener, hereditarer, multisystemischer Bindegewebserkrankungen, die Gelenke, Sehnen, Muskeln, Organe, Haut und Gefase betreffen konnen. Durch die Vielfalt der Symptome und die unterschiedliche Krankheitsauspragung selbst bei gleichem EDS-Typ ist die Diagnostik fur viele Arzte eine Herausforderung. Doch erstmals seit 20 Jahren wurden 2017 eine neue Klassifikation und striktere Kriterien veroffentlicht, die dazu beitragen sollen, dass Patienten schneller diagnostiziert und besser behandelt werden konnen. Laut der neuen Klassifikation differenziert man 13 Typen der Ehlers-Danlos-Syndrome, die sich durch ihre genetischen Ursachen und Verlaufsformen unterscheiden. Am haufigsten kommt das hypermobile EDS (hEDS) vor, welches gleichzeitig in Kombination mit anderen Komorbiditaten wie dem posturalen orthostatischen Tachykardiesyndrom (POTS), dem Mastzellaktivierungssyndrom (MCAS), der Osteoporose, der Neuropathie, der Myopathie, der Mitralklappeninsuffizienz und vielen anderen auftritt. Gerade aufgrund dieser Komorbiditaten ist die Behandlung der EDS schwierig und stellt die verschiedensten Facharzte vor grose Herausforderungen. Neue Managementguidelines widmen sich nicht nur der Behandlung, sondern fokussieren gleichermasen auf die gegenseitige Beeinflussung durch komorbide Erkrankungen und geben einen Leitfaden fur die derzeit nur symptomatische Therapie der EDS. Diese besteht aus konservativer physikalischer Therapie, einer Schmerztherapie, der Nutzung diverser Hilfsmittel, operativen Interventionen und der Behandlung aller komorbiden Faktoren, um die bestmogliche Lebensqualitat zu erzielen. Die Krankheitsauspragung kann von leichter Hypermobilitat bis zu schwerer korperlicher Behinderung reichen.

Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.

Summary The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine., Graphical Abstract, Highlights • G6 binds to a subset of IGHV1-69 germline-based anti-influenza Abs • The structure of humanized G6 with a IGHV1-69 anti-influenza Ab is reported • Various binding assays further define the G6 cross-reactive binding idiotope • The core binding idiotope of G6 is deduced, G6 is an exceptional anti-idiotypic antibody that binds to antibodies encoded by the immunoglobulin heavy chain germline gene IGHV1-69. Avnir et al. describe how G6 binds to its cross-reactive idiotope by a set of binding assays and by solving the structure of humanized-G6 complexed with an IGHV1-69-anti-influenza Ab.

Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi’s sarcoma-associated herpes-virus (KSHV) as a model system, we sought to dissect a putative allosteric network linking a cryptic site at the dimerization interface to enzyme function. Five cryogenic X-ray structures were solved of the monomeric protease with allosteric inhibitors bound to the dimer interface site. Distinct coordinated movements captured by the allosteric inhibitors were also revealed as alternative states in room-temperature X-ray data and comparative analyses of other dimeric herpesvirus proteases. A two-step mechanism was elucidated through detailed kinetic analyses and suggests an enzyme isomerization model of inhibition. Finally, a representative allosteric inhibitor from this class was shown to be efficacious in a cellular model of viral infectivity. These studies reveal a coordinated dynamic network of atomic communication linking cryptic binding site occupancy and allosteric inactivation of KHSV Pr that can be exploited to target other members of this clinically relevant family of enzymes.

Protein-protein interactions between E3 ubiquitin ligases and protein termini shape the proteome. These interactions are sensitive to proteolysis, which alters the ensemble of cellular N- and C-termini. We have identified a mechanism wherein caspase activity reveals latent C-termini that are recognized by the E3 ubiquitin ligase CHIP. We expanded established CHIP specificity and identified hundreds of putative CHIP interactions arising from caspase activity. Subsequent validation confirmed that CHIP binds the latent C-termini at tau(D421) and caspase-6(D179) in vitro and in cells. CHIP binding to tau(D421), but not tau(FL), promotes its ubiquitination, while binding to caspase-6(D179) mediates ubiquitin-independent inhibition. Given that caspase activity generates tau(D421) in Alzheimer’s Disease (AD), these results suggest a concise model for CHIP regulation of tau homeostasis. Indeed, we find that a loss of CHIP expression in AD coincides with the accumulation of tau(D421) and caspase-6(D179). These results illustrate an unanticipated link between caspases and protein homeostasis.

Some variants that cause autosomal-recessive congenital adrenal hyperplasia (CAH) also cause hypermobility type Ehlers-Danlos syndrome (EDS) due to the monoallelic presence of a chimera disrupting two flanking genes: CYP21A2, encoding 21-hydroxylase, necessary for cortisol and aldosterone biosynthesis, and TNXB, encoding tenascin-X, an extracellular matrix protein. Two types of CAH tenascin-X (CAH-X) chimeras have been described with a total deletion of CYP21A2 and characteristic TNXB variants. CAH-X CH-1 has a TNXB exon 35 120-bp deletion resulting in haploinsufficiency, and CAH-X CH-2 has a TNXB exon 40 c.12174C>G (p.Cys4058Trp) variant resulting in a dominant-negative effect. We present here three patients with biallelic CAH-X and identify a novel dominant-negative chimera termed CAH-X CH-3. Compared with monoallelic CAH-X, biallelic CAH-X results in a more severe phenotype with skin features characteristic of classical EDS. We present evidence for disrupted tenascin-X function and computational data linking the type of TNXB variant to disease severity.

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics., Cytidine deaminases are evolutionarily conserved enzymes that edit genomes by deaminating cytidine to uridine. Here the authors present the crystal structure of APOBEC3A with a single-stranded DNA substrate bound in the active site to shed light on the mechanism and specificity of substrate recognition.

SummaryHuman APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.

APOBEC3s (A3) are Zn2+ dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by ‘hypermutating’ the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn2+ coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design.

High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort.REdiiiis a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies in case the preconfigured solution is inadequate. This integrated pipeline is targeted at providing a comprehensive and efficient approach to screening for ligand-bound co-crystal structures while minimizing repetitiveness and allowing a high-throughput scientific discovery process.

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data co...