Noncovalent antibody catenation on a target surface greatly increases the antigen-binding avidity.

Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g. dissociation constant of 100 µM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.

Corrigendum: Structural basis for the broad and potent cross-reactivity of an N501Y-centric antibody against sarbecoviruses.

[This corrects the article DOI: 10.3389/fimmu.2022.1049867.].

Structural basis for the broad and potent cross-reactivity of an N501Y-centric antibody against sarbecoviruses.

More than 80% of SARS-CoV-2 variants, including Alpha and Omicron, contain an N501Y mutation in the receptor-binding domain (RBD) of the spike protein. The N501Y change is an adaptive mutation enabling tighter interaction with the human ACE2 receptor. We have developed a broadly neutralizing antibody (nAb), D27LEY, whose binding affinity was intentionally optimized for Y501. This N501Y-centric antibody not only interacts with the Y501-containing RBDs of SARS-CoV-2 variants, including Omicron, with pico- or subnanomolar binding affinity, but also binds tightly to the RBDs with a different amino acid at residue 501. The crystal structure of the Fab fragment of D27LEY bound to the RBD of the Alpha variant reveals that the Y501-containing loop adopts a ribbon-like topology and serves as a small but major epitope in which Y501 is a part of extensive intermolecular interactions. A hydrophobic cleft on the most conserved surface of the RBD core serves as another major binding epitope. These data explain the broad and potent cross-reactivity of this N501Y-centric antibody, and suggest that a vaccine antigenic component composed of the RBD core and a part of receptor-binding motif (RBM) containing tyrosine at residue 501 might elicit broad and potent humoral responses across sarbecoviruses.

Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently.

One of the hallmarks of cancer cells is their ability to evade apoptosis, which confers survival advantages and resistance to anti-cancer drugs. Cancers often exhibit overexpression of anti-apoptotic BCL-2 proteins, the loss of which triggers apoptosis. In particular, the inhibition of both BCL-xL and MCL-1, but neither one individually, synergistically enhances apoptotic cell death. Here, we report computational design to produce a protein that inhibits both BCL-xL and MCL-1 simultaneously. To a reported artificial three-helix bundle whose second helix was designed to bind MCL-1, we added a fourth helix and designed it to bind BCL-xL. After structural validation of the design and further structure-based sequence design, we produced a dual-binding protein that interacts with both BCL-xL and MCL-1 with apparent dissociation constants of 820 pM and 196 pM, respectively. Expression of this dual binder in a subset of cancer cells induced apoptotic cell death at levels significantly higher than those induced by the pro-apoptotic BIM protein. With a genetic fusion of a mitochondria-targeting sequence or the BH3 sequence of BIM, the activity of the dual binder was enhanced even further. These data suggest that targeted delivery of this dual binder alone or as a part of a modular protein to cancers in the form of protein, mRNA, or DNA may be an effective way to induce cancer cell apoptosis.

Reversible modification of mitochondrial ADP/ATP translocases by paired Legionella effector proteins.

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria's benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.

Functional role of a novel algicidal compound produced by Pseudoruegeria sp. M32A2M on the harmful algae Alexandrium catenella.

A marine phytoplankton dinoflagellate, Alexandrium sp. is known to cause worldwide harmful algal blooms, resulting in paralytic shellfish poisoning. In this study, we isolated a novel compound secreted by the marine bacterium Pseudoruegeria sp. M32A2M, and showed that it displays algicidal activity against A. catenella (group I). The molecular structure of the compound was analyzed by using 1H nuclear magnetic resonance (NMR), 13C NMR, and gas chromatography-mass spectrometry, which revealed that the compound was a diketopiperazine, cyclo[Ala-Gly]. Cyclo[Ala-Gly] induced a rapid decrease in the active chlorophyll a content and maximal quantum yield of photosystem II, leading to membrane disintegration after 24 h of its treatment. It showed the highest algicidal effect against diketopiperazines and also showed specific algicidal activities against several dinoflagellate species, but not for diatom species. In particular, cyclo[Ala-Gly] caused the transcriptional downregulation of the photosynthesis-related membrane complex in A. catenella, but not in the diatom Chaetoceros simplex. Based on structural modeling, we elucidated that cyclo[Ala-Gly] has a structure similar to that of plastoquinone, which transfers electrons by binding to the photosystem II core proteins PsbA and PsbD. This suggests a novel role for cyclo[Ala-Gly] as a potential inhibitor of photosynthesis.

Structural and functional characterization of a monoclonal antibody blocking TIGIT.

TIGIT is an immune checkpoint receptor that is expressed on subsets of activated T cells and natural killer (NK) cells. Several ligands for TIGIT, including poliovirus receptor (PVR), are expressed on cancer cells and mediate inhibitory signaling to suppress antitumor activities of the immune cells. Many studies support that the TIGIT signaling is a potential target for cancer immunotherapy. We developed an IgG4-type monoclonal antibody against human TIGIT, designated as MG1131, using a phage display library of single-chain variable fragments (scFvs). MG1131 interacts with TIGIT much more tightly than PVR does. The crystal structure of a scFv version of MG1131 bound to TIGIT was determined, showing that MG1131 could block the PVR-TIGIT interaction and thus the immunosuppressive signaling of TIGIT. Consistently, MG1131 is bound to TIGIT-expressing cells and interferes with PVR binding to these cells. Moreover, MG1131 increased NK cell-mediated tumor killing activities, inhibited immunosuppressive activity of regulatory T (Treg) cells from healthy donors, and restored interferon-γ secretion from peripheral blood mononuclear cells derived from multiple myeloma patients. MG1131 also increased T cell infiltration to the tumor site and inhibited tumor growth in mice. Collectively, these data indicate that MG1131 modulates the effector functions of T cells and NK cells positively and Treg cells negatively.

Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Dimeric architecture of maltodextrin glucosidase (MalZ) provides insights into the substrate recognition and hydrolysis mechanism.

Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes.

Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.

De novo design of modular and tunable protein biosensors.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Crystal structure of PYCH_01220 from Pyrococcus yayanosii potentially involved in binding nucleic acid.

We report the crystal structure of PYCH_01220, a hypothetical protein in Pyrococcus yayanosii CH1. This protein is composed of two domains, named Domain A and Domain B. While Domain B is not significantly homologous to known protein structures, Domain A is structurally analogous to the C-terminal ribonuclease domain of Escherichia coli colicin D. Domain A has a positively charged surface patch rendered by 13 basic residues, eight arginine or lysine residues of which are evolutionarily conserved. Electrophoretic mobility shift assays showed that PYCH_01220 binds to DNA, and charge-inversion mutations on this patch negatively affect the DNA binding, suggesting that the function of PYCH_01220 might involve nucleic acid-binding via the positively charged patch.

MG1113, a specific anti-tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia.

BACKGROUND: Replacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. OBJECTIVES: To reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti-tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. METHODS: MG1113 is an IgG4 monoclonal antibody that binds to Kunitz-2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)- or IX (FIX)-deficient plasma. For the ex vivo spiking test, platelet-poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)-induced rabbits. RESULTS: Radiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII-deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration-dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113-treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA-induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. CONCLUSIONS: These results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.

De novo design of modular and tunable allosteric biosensors.

Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1 , but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2 . Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3 , we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex.

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.

Author Correction: A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evidence for binary Smc complexes lacking kite subunits in archaea.

SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc-ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc-ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.

Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.

In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens.

Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5' target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.