Construction of dual-cofactor artificial metalloenzymes for synergistic and enantiodivergent catalysis of Michael addition reactions

W. Wang; X. Ji; P. Gorrea-Acín; K. Lau; F. Pojer  et al.

The Vsr-like protein FASTKD4 regulates the stability and polyadenylation of the MT-ND3 mRNA

X. Yang; M. Stentenbach; L. A. Hughes; S. J. Siira; K. Lau  et al.

Expression of the compact mitochondrial genome is regulated by nuclear encoded, mitochondrially localized RNA-binding proteins (RBPs). RBPs regulate the lifecycles of mitochondrial RNAs from transcription to degradation by mediating RNA processing , maturation, st abilit y and translation. T he F as-activ ated serine / threonine kinase (FAS TK) f amily of RBPs has been sho wn to regulate and fine-tune discrete aspects of mitochondrial gene expression. Although the roles of specific targets of FASTK proteins have been elucidated, the molecular mechanisms of FASTK proteins in mitochondrial RNA metabolism remain unclear. T heref ore, w e resolv ed the str uct ure of FA S TKD4 at atomic le v el that includes the RAP domain and the two FAST motifs, creating a positively charged cavity resembling that of the very short patch repair endonuclease. Our biochemical studies show that FASTKD4 binds the canonical poly(A) tail of MT-ND3 enabling its maturation and translation. The in vitro role of FASTKD4 is consistent with its loss in cells that results in decreased MT-ND3 poly aden ylation, which destabilizes this messenger RNA in mitochondria.

Artificial Metalloenzymes with Two Catalytic Cofactors for Tandem Abiotic Transformations

W. Wang; R. Tachibana; K. Zhang; K. Lau; F. Pojer  et al.

Artificial metalloenzymes (ArMs) enable the integration of abiotic cofactors within a native protein scaffold, allowing for non-natural catalytic activities. Previous ArMs, however, have primarily relied on single cofactor systems, limiting them to only one catalytic function. Here we present an approach to construct ArMs embedding two catalytic cofactors based on the biotin-streptavidin technology. By incorporating multiple catalytic cofactors into the four binding sites of streptavidin, we engineered programmable ArMs for tandem abiotic transformations including an enantioselective formal C−H hydroxylation and a photooxidation-Michael addition. This work thus outlines a promising strategy for the development of ArMs embedding multiple cofactors.

Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

N. Thakur; J. Newman; D. Ni; J. Seow; A. L. Hay  et al.

Sarbecoviruses interact with their receptor, angiotensin converting enzyme 2 (ACE2), via the receptor binding domain (RBD) of Spike, the immunodominant target for neutralising antibodies. Understanding the interplay and correlation between ACE2-determined host range and antigenicity is vitally important for understanding the zoonotic potential of related bat sarbecoviruses. Using binding assays, pseudotype-entry assays and a diverse panel of mammalian ACE2 proteins, we examined the host range and related antigenicity of multiple bat coronaviruses. Broad bat ACE2 usage (a generalist phenotype) was most common in clade 1 sarbecoviruses, including SARS-CoV-2 and the BANAL isolates from Laos. In contrast, clade 3 (e.g., RhGB07) and 5 (e.g., Rc-o319) sarbecoviruses exhibited more restricted ACE2 usage (a specialist phenotype). A novel structure for RhGB07 Spike further helped to identify RBD residues associated with this receptor specialism. Interestingly, the generalist phenotypes were largely maintained with more diverse mammalian receptor libraries, including human, non-human primate, livestock, rodent ACE2 and potential intermediate reservoir hosts (e.g., civet, racoon dog, pangolin), while specialists, like RhGB07, exhibited wider phenotypic diversity. The impact of SARS-CoV-2’s continued evolution in humans was also examined, identifying an expanding and/or shifting pattern of generalism for variants, especially Omicron and its sub-lineages. Furthermore, we compared and correlated these entry phenotypes with antigenicity using sera from SARS-CoV-2 convalescent individuals. Clade 1 viruses, phylogenetically related to SARS-CoV-2, were antigenically the most similar, with robust evidence for cross-neutralisation; however, there was still evidence for limited cross-neutralisation across the entire sub-genus. Finally, using monoclonal antibodies, derived from COVID-19 vaccinees with breakthrough infections, we pin-pointed the antibody epitope classes responsible for wider neutralisation. Our research indicates that generalist ACE2-using sarbecoviruses are phylogenetically and antigenically related to SARS-CoV-2.

Hydrophobic tuning with non-canonical amino acids in a copper metalloenzyme

S. Fischer; A. Natter Perdiguero; K. Lau; A. Deliz Liang

Hydrophobicity controls many aspects of protein and enzyme function. Although hydrophobic tuning can be achieved to a limited extent with canonical amino acids, the incorporation of noncanonical amino acids (ncAAs) further extends this ability to enable new and improved functionality. Herein, we engineer an aminoacyl-tRNA synthetase/tRNA pair for the site-specific genetic encoding of a set of bulky, hydrophobic amino acids, namely cyclopentylalanine, cyclohexylalanine, and cycloheptylalanine. With the resulting orthogonal translations systems, we demonstrate the utility of ncAA-based hydrophobic tuning to engineer a bacterial laccase, which is both a classical metalloenzyme and a high-value catalyst for industrial processes. The resulting mutations conveyed significant improvements in catalytic activity, particularly kcat and total turnover number. The redox potential and structure-function relationships were examined to elucidate the source of this improved functionality. We envision that these tools for hydrophobic tuning will be highly valuable for general enzyme engineering and also in other fields, including peptide chemistry.

Purification of SerMNucA (Benzonase/Turbonuclease) as 10xHis, MBP or myc-tag fusions v1

L. Rieubon; K. Lau; F. Pojer

During the lysis of cells, in particular of eukaryotic origin, there is a large release of nucleic acids. The presence of DNA/RNA increases the viscosity of a lysate and may pose problems during downstream processes. Removal of nucleic acids can be performed in many ways, of which one is by enzymatic digestion by nucleases. DNase/RNase are commonly added during protein purification alongside other commercial enzymes such as Benzonase (Novagen) and Turbonuclease. The latter enzymes are industrially produced or recombinant versions of the Serratia marcescens nuclease. This enzyme, hereafter referred to as SerMNucA, is able to hydrolyze both single- and double-stranded DNA and RNA with high activity. Reported methods have shown that the nuclease can be made as inclusion bodies followed by refolding or secreted in the extracellular media. Here we report the purification of the SerMNucA nuclease in a soluble form, followed by purification from either periplasm or cell lysate. We find that purified protein from either fraction are active in a salmon sperm degradation assay with the periplasmic fraction slightly more. This protocol allows for a lab to independently produce tagged and untagged versions of the SerMNucA endonuclease in large quantities that may be required for certain experiments that may be cost prohibitive otherwise. 3 versions are reported here: 10x-His tagged MBP tagged myc tagged The use of the enzyme is in such small quantities that repurification with the tag should not be an issue. The MBP tagged version is cleavable with TEV protease (see also: 10x-His-SuperTEV). Finally the myc-tagged version should not interfere with any common protein purification protocols.

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

B. Küçükoğlu; I. Mohammed; R. C. Guerrero-Ferreira; S. M. Ribet; G. Varnavides  et al.

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography – a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) – has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research

E. J. Mueller; S. Rahimi; P. Sauta; T. Shojaeian; L. Durrer  et al.

Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor–mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. Basic Protocol 1: Bovine IgG stripping from FBS and quality control. Basic Protocol 2: AK23 hybridoma expansion and IgG production. Basic Protocol 3: AK23 IgG purification. Basic Protocol 4: AK23 IgG quality control. Support Protocol 1: Detection of endotoxin levels. Support Protocol 2: Detection and removal of mycoplasma.

Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins

A. S. Petruzzella; M. Bruand; A. Santamaria-Martinez; N. Katanayeva; L. Reymond  et al.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology

W. Wang; R. Tachibana; Z. Zou; D. Chen; X. Zhang  et al.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.|Efficient Mn artificial transfer hydrogenases (ATHases) were developed using the biotin-streptavidin technology, which exhibits high activity and enantioselectivity for the transfer hydrogenation of a wide range of aryl ketones. The S112Y-K121 M double mutation and the appropriate chemical structure of the Mn cofactor play critical roles in the reactivity and enantioselectivity of the enzymes.image

Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

C. C. S. Tan; J. Trew; T. P. Peacock; K. Y. Mok; C. Hart  et al.

There has been limited characterisation of bat-borne coronaviruses in Europe. Here, we screened for coronaviruses in 48 faecal samples from 16 of the 17 bat species breeding in the UK, collected through a bat rehabilitation and conservationist network. We recovered nine complete genomes, including two novel coronavirus species, across six bat species: four alphacoronaviruses, a MERS-related betacoronavirus, and four closely related sarbecoviruses. We demonstrate that at least one of these sarbecoviruses can bind and use the human ACE2 receptor for infecting human cells, albeit suboptimally. Additionally, the spike proteins of these sarbecoviruses possess an R-A-K-Q motif, which lies only one nucleotide mutation away from a furin cleavage site (FCS) that enhances infectivity in other coronaviruses, including SARS-CoV-2. However, mutating this motif to an FCS does not enable spike cleavage. Overall, while UK sarbecoviruses would require further molecular adaptations to infect humans, their zoonotic risk warrants closer surveillance.

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes

J. Racle; P. Guillaume; J. Schmidt; J. Michaux; A. Larabi  et al.

CD4+T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our under-standing of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specific-ities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes

J. Racle; P. Guillaume; J. Schmidt; J. Michaux; A. Larabi  et al.

CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.

High-affinity peptides developed against calprotectin and their application as synthetic ligands in diagnostic assays

C. Diaz-Perlas; B. Ricken; L. Farrera-Soler; D. Guschin; F. Pojer  et al.

A peptide was developed that binds to calprotectin, a marker of major inflammatory disorders, and found to be suited for diagnostic tests. The use of synthetic peptides in assays is of great interest due to their high precision, robustness and low price.

Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain

L. Dumas; M. Marfoglia; B. Yang; M. Hijazi; A. Larabi  et al.

Key cellular functions depend on the transduction of extracellular mechanical signals by specialized membrane receptors including adhesion G-protein coupled receptors (aGPCRs). While recently solved structures support aGPCR activation through shedding of the extracellular GAIN domain, the molecular mechanisms underpinning receptor mechanosensing remain poorly understood. When probed using single-molecule atomic force spectroscopy and molecular simulations, ADGRG1 GAIN dissociated from its tethered agonist at forces significantly higher than other reported signaling mechanoreceptors. Strong mechanical resistance was achieved through specific structural deformations and force propagation pathways under mechanical load. ADGRG1 GAIN variants computationally designed to lock the alpha and beta subdomains and rewire mechanically-induced structural deformations were found to modulate the GPS-Stachel rupture forces. Our study provides unprecedented insights into the molecular underpinnings of GAIN mechanical stability and paves the way for engineering mechanosensors, better understanding aGPCR function, and informing drug-discovery efforts targeting this important receptor class.

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

D. Ni; P. Turelli; B. Beckert; S. Nazarov; E. Uchikawa  et al.

nvestigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern

D. Ni; P. Turelli; B. Beckert; S. Nazarov; E. Uchikawa  et al.

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

Broadly potent anti-SARS-CoV-2 antibody shares 93% of epitope with ACE2 and provides full protection in monkeys

C. Fenwick; P. Turelli; Y. Duhoo; K. Lau; C. Herate  et al.

Due to the rapid evolution of SARS-CoV-2 to variants with reduced sensitivity to vaccine-induced humoral immunity and the near complete loss of protective efficacy of licensed therapeutic monoclonal antibodies, we isolated a potent, broad-spectrum neutralizing antibody that could potentially provide prophylactic protection to immunocompromised patient populations.

ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys

C. Fenwick; P. Turelli; Y. Duhoo; K. Lau; C. Herate  et al.

The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares ∼93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding ‘escape’ mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.

Purification of 10xHis-SuperTEV v1

K. Lau; B. Bouchri; F. Pojer

SuperTEV is a mutated version of TEV (Tobacco Etch Protease) a cysteine protease widely used in labs as it is highly specific to a cleavage sequence that can be genetically encoded. Depending on the lab, it has been reported that the protease is unstable or purifies with low yields. Efforts have been made for many years (Tropea, 2009, Methods Mol Biol) to improve its solubility. Here we report our platform's efforts in generating a new TEV variant, called SuperTEV, that incorporates 9 mutations identified by different groups in recent years applied together at once. We find that the protein is easily produced and purified in large amounts and is functional. The list of mutations on the canonical TEV protease are the following (with references that also identified the same mutations): T17S (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 L56V (rational design, improved solubility) Cabrita et al., 2007 and Wei et al., 2012 N68D (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 I77V (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012 S135G (rational design, increased solubility) Cabrita et al., 2007 and Wei et al., 2012 I138T (increased catalytic activity; TEV3) Sanchez and Ting, 2019 S153N (increased catalytic activity; TEV3) Sanchez and Ting, 2019 T180A (increased catalytic activity; TEV3) Sanchez and Ting, 2019 S219V (inhibits autoproteolysis) Kapust, 2001