Experimental Structures of Antibody/MHC-I Complexes Reveal Details of Epitopes Overlooked by Computational Prediction.

mAbs to MHC class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I Fabs bound to peptide/MHC-I/ß2-microglobulin (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAbs, and an anti-ß2-microglobulin mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

Molecules that Generate Fingerprints: A New Class of Fluorescent Sensors for Chemical Biology, Medical Diagnosis, and Cryptography.

Fluorescent molecular sensors, often referred to as "turn-on" or "turn-off" fluorescent probes, are synthetic agents that change their fluorescence signal in response to analyte binding. Although these sensors have become powerful analytical tools in a wide range of research fields, they are generally limited to detecting only one or a few analytes. Pattern-generating fluorescent probes, which can generate unique identification (ID) fingerprints for different analytes, have recently emerged as a new class of luminescent sensors that can address this limitation. A unique characteristic of these probes, termed ID-probes, is that they integrate the qualities of conventional small-molecule-based fluorescent sensors and cross-reactive sensor arrays (often referred to as chemical, optical, or electronic noses/tongues). On the one hand, ID-probes can discriminate between various analytes and their combinations, akin to array-based analytical devices. On the other hand, their minute size enables them to analyze small-volume samples, track dynamic changes in a single solution, and operate in the microscopic world, which the macroscopic arrays cannot access.Here, we describe the principles underlying the ID-probe technology, as well as provide an overview of different ID-probes that have been developed to date and the ways they can be applied to a wide range of research fields. We describe, for example, ID-probes that can identify combinations of protein biomarkers in biofluids and in living cells, screen for several protein inhibitors simultaneously, analyze the content of Aß aggregates, as well as ensure the quality of small-molecule and biological drugs. These examples highlight the relevance of this technology to medical diagnosis, bioassay development, cell and chemical biology, and pharmaceutical quality assurance, among others. ID-probes that can authorize users and protect secret data are also presented and the mechanisms that enable them to hide (steganography), encrypt (cryptography), and prevent access to (password protection) information are discussed.The versatility of this technology is further demonstrated by describing two types of probes: unimolecular ID-probes and self-assembled ID-probes. Probes from the first type can operate inside living cells, be recycled, and their initial patterns can be more easily obtained in a reproducible manner. The second type of probes can be readily modified and optimized, allowing one to prepare various different probes from a much wider range of fluorescent reporters and supramolecular recognition elements. Taken together, these developments indicate that the ID-probe sensing methodology is generally applicable, and that such probes can better characterize analyte mixtures or process chemically encoded information than can the conventional fluorescent molecular sensors. We therefore hope that this review will inspire the development of new types of pattern-generating probes, which would extend the fluorescence molecular toolbox currently used in the analytical sciences.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

Applications of Bacteria Decorated with Synthetic DNA Constructs.

The advent of DNA nanotechnology has revolutionized the way DNA has been perceived. Rather than considering it as the genetic material alone, DNA has emerged as a versatile synthetic scaffold that can be used to create a variety of molecular architectures. Modifying such self-assembled structures with bio-molecular recognition elements has further expanded the scope of DNA nanotechnology, opening up avenues for using synthetic DNA assemblies to sense or regulate biological molecules. Recent advancements in this field have lead to the creation of DNA structures that can be used to modify bacterial cell surfaces and endow the bacteria with new properties. This mini-review focuses on the ways by which synthetic modification of bacterial cell surfaces with DNA constructs can expand the natural functions of bacteria, enabling their potential use in various fields such as material engineering, bio-sensing, and therapy. The challenges and prospects for future advancements in this field are also discussed.

Fluorescent Investigation of Proteins Using DNA-Synthetic Ligand Conjugates.

The unfathomable role that fluorescence detection plays in the life sciences has prompted the development of countless fluorescent labels, sensors, and analytical techniques that can be used to detect and image proteins or investigate their properties. Motivated by the demand for simple-to-produce, modular, and versatile fluorescent tools to study proteins, many research groups have harnessed the advantages of oligodeoxynucleotides (ODNs) for scaffolding such probes. Tight control over the valency and position of protein binders and fluorescent dyes decorating the polynucleotide chain and the ability to predict molecular architectures through self-assembly, inherent solubility, and stability are, in a nutshell, the important properties of DNA probes. This paper reviews the progress in developing DNA-based, fluorescent sensors or labels that navigate toward their protein targets through small-molecule (SM) or peptide ligands. By describing the design, operating principles, and applications of such systems, we aim to highlight the versatility and modularity of this approach and the ability to use ODN-SM or ODN-peptide conjugates for various applications such as protein modification, labeling, and imaging, as well as for biomarker detection, protein surface characterization, and the investigation of multivalency.

Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

An Optical Probe for Real-Time Monitoring of Self-Replicator Emergence and Distinguishing between Replicators.

Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, we now developed a pattern-generating fluorescent molecular probe (an ID-probe) capable of discriminating replicators of different chemical composition and monitoring the process of replicator formation in real time, giving distinct signatures for starting materials, intermediates, and final products. Optical monitoring of replicators dramatically reduces the analysis time and sample quantities compared to most currently used methods and opens the door for future high-throughput experimentation in protocell environments.

Cutting Edge: Inhibition of the Interaction of NK Inhibitory Receptors with MHC Class I Augments Antiviral and Antitumor Immunity.

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

Structural aspects of chaperone-mediated peptide loading in the MHC-I antigen presentation pathway.

Recognition of foreign and dysregulated antigens by the cellular innate and adaptive immune systems is in large part dependent on the cell surface display of peptide/MHC (pMHC) complexes. The formation of such complexes requires the generation of antigenic peptides, proper folding of MHC molecules, loading of peptides onto MHC molecules, glycosylation, and transport to the plasma membrane. This complex series of biosynthetic, biochemical, and cell biological reactions is known as "antigen processing and presentation". Here, we summarize recent work, focused on the structural and functional characterization of the key MHC-I-dedicated chaperones, tapasin, and TAPBPR. The mechanisms reflect the ability of conformationally flexible molecules to adapt to their ligands, and are comparable to similar processes that are exploited in peptide antigen loading in the MHC-II pathway.

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions.

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.

Cutting antigenic peptides down to size.

A critical step in antigen presentation is the degradative processing of peptides by aminopeptidases in the endoplasmic reticulum. It is unclear whether these enzymes act only on free peptides or on those bound to their major histocompatibility complex (MHC)-I-presenting molecules. A recent study examined the structure and biophysics of N-terminally extended peptides in complex with MHC-I, revealing the conformational adjustment of MHC to permit both binding of the peptide core and exposure of the peptide N terminus. These data suggest a mechanism by which aminopeptidase access is determined and offer an explanation for how longer peptides may be displayed at the cell surface.

Glycoform Differentiation by a Targeted, Self-Assembled, Pattern-Generating Protein Surface Sensor.

A method for generating targeted, pattern-generating, protein surface sensors via the self-assembly of modified oligodeoxynucleotides (ODNs) is described. The simplicity by which these systems can be created enabled the development of a sensor that can straightforwardly discriminate between distinct glycoform populations. By using this sensor to identify glycosylation states of a therapeutic protein, we demonstrate the diagnostic potential of this approach as well as the feasibility of integrating a wealth of supramolecular receptors and sensors into higher-order molecular analytical devices with advanced properties. For example, the facile device integration was used to attach the well-known anthracene-boronic acid (An-BA) probe to a biomimetic DNA scaffold and consequently, to use the unique photophysical properties of An-BA to improve glycoform differentiation. In addition, the noncovalent assembly enabled us to modify the sensor with a trinitrilotriacetic acid (tri-NTA)-Ni2+ complex, which endows it with selectivity toward a hexa-histidine tag (His-tag). The selective responses of the system to diverse His-tag-labeled proteins further demonstrate the potential applicability of such sensors and validate the mechanism underlying their function.

MHC-restricted Ag85B-specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Peptide exchange on MHC-I by TAPBPR is driven by a negative allostery release cycle.

Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex.

Effects of Cross-Presentation, Antigen Processing, and Peptide Binding in HIV Evasion of T Cell Immunity.

Unlike cytosolic processing and presentation of viral Ags by virus-infected cells, Ags first expressed in infected nonprofessional APCs, such as CD4+ T cells in the case of HIV, are taken up by dendritic cells and cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases, such as cathepsin S (CatS). We have modified HIV-1MN gp120 by mutating a key CatS cleavage site (Thr322Thr323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation and provide evidence from MHC binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the MHC class I molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild-type protein is immunogenic without this mutation. These proof-of-concept results show that a virus like HIV, infecting predominantly nonprofessional presenting cells, can escape T cell recognition by incorporating a CatS cleavage site that leads to destruction of an immunodominant epitope when the Ag undergoes endosomal cross-presentation.

Structure and Function of Molecular Chaperones that Govern Immune Peptide Loading.

Major histocompatibility class I (MHC-I) molecules bind peptides derived from cellular synthesis and display them at the cell surface for recognition by receptors on T lymphocytes (TCR) or natural killer (NK) cells. Such recognition provides a crucial step in autoimmunity, identification of bacterial and viral pathogens, and anti-tumor responses. Understanding the mechanism by which such antigenic peptides in the ER are loaded and exchanged for higher affinity peptides onto MHC molecules has recently been clarified by cryo-EM and X-ray studies of the multimolecular peptide loading complex (PLC) and a unimolecular tapasin-like chaperone designated TAPBPR. Insights from these structural studies and complementary solution NMR experiments provide a basis for understanding mechanisms related to immune antigen presentation.

Encrypting messages with artificial bacterial receptors.

A method for encrypting messages using engineered bacteria and different fluorescently labeled synthetic receptors is described. We show that the binding of DNA-based artificial receptors to E. coli expressing His-tagged outer membrane protein C (His-OmpC) induces a Förster resonance energy transfer (FRET) between the dyes, which results in the generation of a unique fluorescence fingerprint. Because the bacteria continuously divide, the emission pattern generated by the modified bacteria dynamically changes, enabling the system to produce encryption keys that change with time. Thus, this development indicates the potential contribution of live-cell-based encryption systems to the emerging area of information protection at the molecular level.