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Nanobodies (Nbs) have shown great potential in immunodetection of small-molecule contaminants in food and environmental monitoring. However, the limited knowledge of the mechanism of Nbs binding to small molecules has hampered the development of high-affinity Nbs and assay improvement. We previously reported two homologous nanobodies Nb26 and Nb28 specific to aflatoxin B1 (AFB1), with the former exhibiting higher sensitivity in ELISA. Herein, Nb26 was selected as the model antibody to resolve its solution nuclear magnetic resonance (NMR) structure, and investigate its AFB1 recognition mechanism. The results revealed that Nb26 exhibits a typical immunoglobulin fold and its AFB1-binding interface is uniquely located in complementarity-determining region 3 (CDR3) and framework region 2 (FR2). This finding was applied to improve the binding activity of Nb28 against AFB1 by constructing two Nb28-based mutants A50V and S102D, resulting in 2.3- and 3.3-fold sensitivity enhancement over the wild type, respectively. This study develops an NMR-based strategy to analyze the underlying mechanism of Nb against AFB1, and successfully generated two site-modified Nbs with improved detection sensitivity. It is believed that this work could greatly expand the applications of Nbs by providing a way to enhance the binding activity.

Circadian rhythms in skeletal muscle are maintained by a transcriptional-translational feedback loop known as the molecular clock. While previous research suggested a role for the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomeric proteins. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we show that deletion of the skeletal muscle molecular clock alters titin isoform and skeletal muscle sarcomere length. We then use U7 snRNPs in myotubes to directly alter titin splicing in vitro. Truncating titin’s proximal Ig domain results in altered sarcomere length. Finally, we identify a mechanism whereby the skeletal muscle molecular clock regulates titin isoform expression through RBM20, a potent splicing regulator of the titin transcript. Our findings demonstrate the importance of the skeletal muscle molecular clock in maintaining sarcomere length homogeneity through its regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many diseases, our results highlight a pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies. Significance Statement Circadian rhythms regulate many aspects of skeletal muscle physiology; however, the exact mechanisms connecting the molecular underpinnings of these rhythms to skeletal muscle structure and function are poorly understood. Here we describe how the skeletal muscle molecular clock regulates titin, the protein ruler regulating sarcomere length and muscle strength. Since circadian rhythms and skeletal muscle weakness underly a number of diseases, these results highlight a potential target for future therapeutic strategies.

Bovine butyrophilin (BTN1A1) is an abundant type I transmembrane glycoprotein exposed on the surface of milk fat globules. We have solved the crystal structure of its extracellular region via multiple wavelength anomalous dispersion after incorporation of selenomethionine into the bacterially produced protein. The butyrophilin ectodomain exhibits two subdomains with immunoglobulin fold, each comprising a β-sandwich with a central disulfide bridge as well as one N-linked glycosylation. The fifth Cys residue at position 193 is unpaired and prone to forming disulfide crosslinks. The apparent lack of a ligand-binding site or receptor activity suggests a function predominantly as hydrophilic coat protein to prevent coagulation of the milk fat droplets. While there is less structural resemblance to members of the human butyrophilin family such as BTN3A, which play a role as immune receptors, the N-terminal bovine butyrophilin subdomain shows surprising similarity to the human myelin oligodendrocyte glycoprotein, a protein exposed on the surface of myelin sheaths. Thus, our study lends structural support to earlier hypotheses of a correlation between the consumption of cow milk and prevalence of neurological autoimmune diseases and may offer guidance for the breeding of cattle strains that express modified butyrophilin showing less immunological cross-reactivity.

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.

Pathogen-derived peptides are loaded on MHC class II (MHCII) and presented to CD4+ T cells for their activation. Peptide loading of MHCII occurs in specialized endosomal compartments and is controlled by the nonclassical MHCII molecules H2-M and H2-O, which are both constitutive αβ heterodimers. H2-M catalyzes MHCII peptide loading, whereas H2-O modulates H2-M activity by acting as an MHCII mimic. Recently, we discovered that the H2-Ob allele inherited by retrovirus-resistant I/LnJ mice results in nonfunctional H2-O. I/LnJ H2-O binds to but does not inhibit H2-M. Compared with H2-Oβ from virus-susceptible mice, H2-Oβ from I/LnJ mice has four unique amino acid substitutions, three in the Ig domain and one in the cytoplasmic tail. In this study we show that the three amino acids in the Ig domain of I/LnJ Oβ are critical for the H2-O inhibitory activity of H2-M. Unexpectedly, we found that MHCII presentation was significantly different in Ag-presenting cells from two closely related mouse strains, B6J and B6N, which carry identical alleles of MHCII, H2-O, and H2-M. Using a positional cloning approach, we have identified two loci, polymorphic between B6J and B6N, that mediate the difference in MHCII presentation. Collectively, these studies reveal extra complexity in MHCII/H2-M/H-2O interactions that likely involve yet to be identified modulators of the pathway.

Receptors are important pharmacological targets on cells. The Triggering Receptor Expressed on Myeloid Cells (TREM) – Like Transcript – 1 is an abundant, yet little understood, platelet receptor. It is a single Ig domain containing receptor isolated in the α-granules of resting platelets and brought to the platelet surface upon activation. On platelets, the integrin αIIbβ3 is the major receptor having roughly 80,000 copies. αIIbβ3 is a heterodimeric multidomain structure that mediates platelet aggregation through its interaction with the plasma protein fibrinogen. Anti-platelet drugs have successfully targeted αIIbβ3 to control thrombosis. Like αIIbβ3, TLT-1 also binds fibrinogen, making its role in platelet function somewhat obscure. In this review, we highlight the known structural features of TLT-1 and present the challenges of understanding TLT-1 function. In our analysis of the dynamics of the platelet surface after activation we propose a model in which TLT-1 supports αIIbβ3 function as a mechanoreceptor that may direct platelets toward immune function.

The IgG CH2 domain continues to hold promise for the development of new therapeutic entities because of its bifunctional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody (mAb) fragments: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to CH2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 °C for all three proteins in an array. Co-distribution and two-dimensional infrared correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, β-sheets, β-turns, and random coil regions were less stable than in CH2s and m01s because of the presence of the N-terminal 310-helix and β-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.

T cell costimulation is mediated by the interaction of a number of receptors and ligands present on the surface of the T cell and antigen-presenting cell, respectively. Stimulatory or inhibitory signals from these receptor-ligand interactions work in tandem to preserve immune homeostasis. BTNL2 is a type-1 membrane protein that provides inhibitory signal to T cells and plays an important role in several inflammatory and autoimmune diseases. Therefore, manipulation of the molecular interaction of BTNL2 with its putative receptor could provide strategies to restore immune homeostasis in these diseases. Hence, it is imperative to study the structural characteristics of this molecule, which will provide important insights into its function as well. In this study, the membrane-distal ectodomain of murine BTNL2 was expressed in bacteria as inclusion bodies, refolded in vitro and purified for functional and structural characterization. The domain is monomeric in solution as demonstrated by size-exclusion chromatography and analytical ultracentrifugation, and also binds to its putative receptor on naïve B cells and activated T cell subsets. Importantly, for the first time, we report the structure of BTNL2 as determined by solution NMR spectroscopy and also the picosecond-nanosecond timescale backbone dynamics of this domain. The N-terminal ectodomain of BTNL2, which was able to inhibit T cell function as well, exhibits distinctive structural features. The N-terminal ectodomain of BTNL2 has a significantly reduced surface area in the front sheet due to the non-canonical conformation of the CC' loop, which provides important insights into the recognition of its presently unknown binding partner.

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. We report that Chelicerata sDscamα and some sDscamβ protein trans interactions are strictly homophilic, and that the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. We report that cell–cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, our data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. We propose that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self–nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, our results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, our findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self–nonself discrimination.

Significance Titin oxidation alters titin stiffness, which greatly contributes to overall myocardial stiffness. This stiffness is frequently increased in heart disease, such as diastolic heart failure. We have quantified the degree of oxidative titin changes in several murine heart and skeletal muscle models exposed to oxidant stress and mechanical load. Importantly, strain enhances in vivo oxidation of titin in the elastic region, but not the inextensible segment. The functional consequences include oxidation type-dependent effects on cardiomyocyte stiffness, titin-domain folding, phosphorylation, and inter-titin interactions. Thus, oxidative modifications stabilize the titin spring in a dynamic and reversible manner and help propagate changes in titin-based myocardial stiffness. Our findings pave the way for interventions that target the pathological stiffness of titin in disease., The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats., Mechanically stable specific heterodimerization formed with reversible bonds are used as a molecular anchorage in single-molecule force spectroscopy studies with unique mechanical properties. Here authors develop a variety of heterodimerization molecular systems with a range of mechanical stability from a set of recently engineered helix-heterotetramers.

The Coxsackie- and adenovirus receptor (CAR) mediates homophilic cell-cell contacts and susceptibility to both human pathogenic viruses through its membrane-distal immunoglobulin domain. In the present study, we screened five missense variants of the human CAR gene for their influence on adenovector or Coxsackievirus entry into Chinese hamster ovary cells. The CAR variants facilitated virus internalisation to a similar extent as wild type CAR. This underlines CAR's presumed invariance and essential physiological role in embryogenesis. Copyright © 2020 Elsevier Inc. All rights reserved.

The traces of immunoglobulin domain similarities were searched in sequences of higher plants using bioinformatic tools to look for possible early phylogenic structural relationships. 280 thousand sequence IDs, obtained by sixteen types of primary BLAST searches, were differently processed by seventeen selection procedures and an anti-redundant sequence-related approach using JavaScript, PHP, Windows programs and conserved domain searches by means CDD. The resulting seventeen sets of records describing conserved domain similarities of 1323 different sequence IDs yielded a set of next generation (final set) comprising forty-nine records containing superior (“non-refutable”) conserved immunoglobulin domain similarities. The selected sets and their subsets were mapped and subsequently statistically compared with respect to immunoglobulin-related as well as other reciprocal domain linkages. The list of frequently occurring conserved domain similarities concerned first of all domains important for plant and metazoan immunity, e.g. tyrosine kinases accompanying variable immunoglobulin domains in early Metazoa, toll-like receptors, lectin and leucine-rich repeat domains. Detailed description of immunoglobulin domain similarities occurring in the final set was completed by fold analysis of the restricted segments. The data were then discussed with respect to i) immunoglobulin fold evolution, ii) possible structural importance of domains cd14066 (IRAK) and PLN00113 (LRR-associated kinase) for deep evolution of catalytic serine/threonine/tyrosine kinase domains, iii) interatomic, structural and specificity standpoints and iv) traces of antibody-like phosphorylation sites described in our previous paper.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

Summary Many prokaryotic cells are covered by an ordered, proteinaceous, sheet-like structure called a surface layer (S-layer). S-layer proteins (SLPs) are usually the highest copy number macromolecules in prokaryotes, playing critical roles in cellular physiology such as blocking predators, scaffolding membranes, and facilitating environmental interactions. Using electron cryomicroscopy of two-dimensional sheets, we report the atomic structure of the S-layer from the archaeal model organism Haloferax volcanii. This S-layer consists of a hexagonal array of tightly interacting immunoglobulin-like domains, which are also found in SLPs across several classes of archaea. Cellular tomography reveal that the S-layer is nearly continuous on the cell surface, completed by pentameric defects in the hexagonal lattice. We further report the atomic structure of the SLP pentamer, which shows markedly different relative arrangements of SLP domains needed to complete the S-layer. Our structural data provide a framework for understanding cell surfaces of archaea at the atomic level., Graphical abstract, Highlights • Cryo-EM reveals the structure of the hexameric S-layer of Haloferax volcanii archaea • Immunoglobulin-like domains are widely conserved in archaeal S-layers • Cryo-ET shows how an S-layer made of hexamers and pentamers coats curved membranes • Atomic-level description of a native archaeal cell surface is shown, von Kügelgen et al. show, using in situ cryo-EM, how Haloferax volcanii archaea are nearly perfectly coated by a hexameric surface layer with pentameric defects, made of a protein with repeated immunoglobulin-like domains. Structure- and sequence-based bioinformatics reveal widespread prokaryotic conservation of these domains, with implications on evolution and fundamental archaeal biology.

A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different variants; some of them may have an impact on the conformational stability of the protein. Conventionally, to evaluate the impact of a variant on stability, the variant must be enriched to a reasonable purity, and then its stability characterized by chromatographic or biophysical techniques. However, it is often impractical to purify and characterize each variant in a therapeutic protein. A workflow, based on limited proteolysis followed by MS detection, was established to simultaneously assess the impact of a large number of variants on conformational stability without enrichment. Because a less stable domain is more susceptible to proteolytic degradation, conformational stability of the domain can be reported from the release rate of a proteolytic peptide. A kinetic model is established to quantitatively determine the extent of domain stabilization/destabilization of different variants. The methodology is demonstrated by examining variants known to affect the stability of immunoglobulin domains, such as different N-glycoforms, methionine oxidations, and sequence variants. With this methodology, near 100 variants may be evaluated within 2 days in a single experiment. Insights into the sequence-stability relationship will be obtained by monitoring the large number of low-level sequence variants, facilitating engineering of more stable molecules.

Molecular mechanisms involved in biological conflicts and self vs nonself recognition in archaea remain poorly characterized. We apply phylogenomic analysis to identify a hypervariable gene module that is widespread among Thermococcales. These loci consist of an upstream gene coding for a large protein containing several immunoglobulin (Ig) domains and unique combinations of downstream genes, some of which also contain Ig domains. In the large Ig domain containing protein, the C-terminal Ig domain sequence is hypervariable, apparently, as a result of recombination between genes from different Thermococcales. To reflect the hypervariability, we denote this gene module VARTIG (VARiable Thermococcales IG). The overall organization of the VARTIG modules is similar to the organization of Polymorphic Toxin Systems (PTS). Archaeal genomes outside Thermococcales encode a variety of Ig domain proteins, but no counterparts to VARTIG and no Ig domains with comparable levels of variability. The specific functions of VARTIG remain unknown but the identified features of this system imply three testable hypotheses: (i) involvement in inter-microbial conflicts analogous to PTS, (ii) role in innate immunity analogous to the vertebrate complement system, and (iii) function in self vs nonself discrimination analogous to the vertebrate Major Histocompatibility Complex. The latter two hypotheses seem to be of particular interest given the apparent analogy to the vertebrate immunity.

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide-bridged form, without forming a disulfide-bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide-bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide-bridge formation. We conclude that, since CH2 can be purified as a folded, semi-stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies.

The Ig-fold has had a remarkable success in vertebrate evolution, with a presence in over 2% of human genes. The Ig-fold is not just the elementary structural domain of antibodies and TCRs, it is also at the heart of a staggering 30% of immunologic cell surface receptors, making it a major orchestrator of cell-cell-interactions. While BCRs, TCRs, and numerous Ig-based cell surface receptors form homo or heterodimers on the same cell surface (in cis), many of them interface as ligand-receptors (checkpoints) on interacting cells (in trans) through their Ig domains. New Ig-Ig interfaces are still being discovered between Ig-based cell surface receptors, even in well known families such as B7. What is largely ignored however is that the Ig-fold itself is pseudo-symmetric, a property that makes the Ig-domain a versatile self-associative 3D structure and may in part explain its success in evolution, especially through its ability to bind in cis or in trans in the context of cell surface receptor-ligand interactions. In this paper we review the Ig domains tertiary and quaternary pseudo symmetries, with a particular attention to the newly identified double Ig fold in the solved CD19 molecular structure to highlight the underlying fundamental folding elements of Ig domains, i.e. Ig protodomains. This pseudosymmetric property of Ig domains gives us a decoding frame of reference to understand the fold, relate all Ig-domain forms, single or double, and suggest new protein engineering avenues.

Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.

T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nm affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Targeting sodium channels Voltage-gated sodium (Na v ) channels have been implicated in cardiac and neurological disorders. There are many subtypes of these channels, making it challenging to develop specific therapeutics. A core α subunit is sufficient for voltage sensing and ion conductance, but function is modulated by β subunits and by natural toxins that can either act as pore blockers or gating modifiers (see the Perspective by Chowdhury and Chanda). Shen et al. present the structures of Na v 1.7 in complex with both β1 and β2 subunits and with animal toxins. Pan et al. present the structure of Na v 1.2 bound to β2 and a toxic peptide, the µ-conotoxin KIIIA. The structure shows why KIIIA is specific for Na v 1.2. These and other recently determined Na v structures provide a framework for targeted drug development. Science , this issue p. 1303 , p. 1309 ; see also p. 1278

Aims Heart failure (HF) patients commonly experience symptoms primarily during elevated heart rates, as a result of physical activities or stress. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with HF, with unresolved involvement regarding its role at different heart rates. To determine whether titin is playing a role in the heart rate (frequency-) dependent acceleration of relaxation (FDAR). W, we studied the FDAR responses in live human left ventricular cardiomyocytes and the corresponding titin-based passive tension (TPT) from failing and non-failing human hearts. Methods and results Using atomic force, we developed a novel single-molecule force spectroscopy approach to detect TPT based on the frequency-modulated cardiac cycle. Mean TPT reduced upon an increased heart rate in non-failing human hearts, while this reduction was significantly blunted in failing human hearts. These mechanical changes in the titin distal Ig domain significantly correlated with the frequency-dependent relaxation kinetics of human cardiomyocytes obtained from the corresponding hearts. Furthermore, the data suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but the lower the potential of myocytes to speed up relaxation at a higher heart rate. Such poorer FDAR response was also associated with a lesser reduction or a bigger increase in TPT upon elevated heart rate. Conclusions Our study established a novel approach in detecting dynamic heart rate relevant tension changes physiologically on native titin domains. Using this approach, the data suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes were associated with the intensity and dynamic changes of passive tension by titin.

Systemic light-chain amyloidosis (AL) is a human disease caused by overexpression of monoclonal immunoglobulin light chains that form pathogenic amyloid fibrils. These amyloid fibrils deposit in tissues and cause organ failure. Proteins form amyloid fibrils when they partly or fully unfold and expose segments capable of stacking into β-sheets that pair and thereby form a tight, dehydrated interface. These structures, termed steric zippers, constitute the spines of amyloid fibrils. Here, using a combination of computational (with ZipperDB and Boston University ALBase), mutational, biochemical, and protein structural analyses, we identified segments within the variable domains of Ig light chains that drive the assembly of amyloid fibrils in AL. We demonstrate that there are at least two such segments and that each one can drive amyloid fibril assembly independently of the other. Our analysis revealed that peptides derived from these segments form steric zippers featuring a typical dry interface with high-surface complementarity and occupy the same spatial location of the Greek-key immunoglobulin fold in both λ and κ variable domains. Of note, some predicted steric-zipper segments did not form amyloid fibrils or assembled into fibrils only when removed from the whole protein. We conclude that steric-zipper propensity must be experimentally validated and that the two segments identified here may represent therapeutic targets. In addition to elucidating the molecular pathogenesis of AL, these findings also provide an experimental approach for identifying segments that drive fibril formation in other amyloid diseases.

Synaptic remodeling plays important roles in health and neural disorders. Although previous studies revealed that several transcriptional programs control synaptic remodeling in the nematode Caenorhabditis elegans, the molecular mechanisms of the dorsal D-type (DD) synaptic remodeling are poorly understood. Here we show that extracellular matrix molecule muscle arm development defective protein-4 (MADD-4) cooperates with the one immunoglobulin domain protein-1 (OIG-1) to defer precocious DD synaptic remodeling. Specifically, loss of MADD-4 exhibited the precocious DD synaptic remodeling. The long isoform MADD-4L is dynamically expressed while the short isoform MADD-4B is persistently expressed in DD neurons of L1 stage. In the unc-30 mutant lacking the Pitx-type homeodomain transcription factor UNC-30, the expression levels of both MADD-4B and -L isoforms were dramatically downregulated in DD neurons of the L1 stage. Our further data showed that MADD-4B and -L isoforms physically interact with OIG-1 and madd-4 acts in the oig-1 genetic pathway to modulate the DD synaptic remodeling. Our findings demonstrated that the extracellular matrix plays a novel role in synaptic plasticity.

Rab18 and V-set and immunoglobulin domain-containing 4 (VSIG4) were reportedly implicated in the malignant progression of glioma. In this study, their relationship was further explored, accompanied by the investigation into their effects on the sensitivity of temozolomide (TMZ). The proliferation and apoptosis of U87-MG and U251-MG were detected after Rab18 silencing through CCK8 assay and flow cytometry, respectively. The interaction between Rab18 and VSIG4 was predicted through database and verified by immunoprecipitation assay. The suspicion that whether the sensitivity of glioma to temozolomide was affected by the Rab18-VSIG4 interaction was explored through CCK8 assay. We observed decreased proliferation and increased apoptosis and TMZ sensitivity in U87-MG and U251-MG treated by siRNA-Rab18. Not only was the interaction predicted using database, but also it was confirmed by IP assay. Intriguingly, VSIG4 overexpression effectively reversed above biological process and TMZ sensitivity caused by Rab18 silencing. To conclude, the Rab18-VSIG4 interaction was implicated in the proliferation and apoptosis of glioma, as well as TMZ sensitivity. Targeting the interaction between Rab18 and VSIG4 may help exploit new therapies to enhance TMZ sensitivity for treating patients with glioma., Graphical abstract

Titin, the largest single chain protein known so far, has long been known to play a critical role in passive muscle function but recent studies have highlighted titin’s role in active muscle function. One of the key elements in this role is the Ca(2+)-dependent interaction between titin’s N2A region and the thin filament. An important element in this interaction is I83, the terminal immunoglobulin domain in the N2A region. There is limited structural information about this domain, but experimental evidence suggests that it plays a critical role in the N2A-actin binding interaction. We now report the solution NMR structure of I83 and characterize its dynamics and metal binding properties in detail. Its structure shows interesting relationships to other I-band Ig domains. Metal binding and dynamics data point towards the way the domain is evolutionarily optimized to interact with neighbouring domains. We also identify a calcium binding site on the N-terminal side of I83, which is expected to impact the interdomain interaction with the I82 domain. Together these results provide a first step towards a better understanding of the physiological effects associated with deletion of most of the I83 domain, as occurs in the mdm mouse model, as well as for future investigations of the N2A region.