Ligand Recognition Domain Research Articles

Intrinsic Disorder in Nuclear Receptor Amino Termini: From Investigational Challenge to Therapeutic Opportunity

Epidermal keratinocytes form an effective renewable barrier to surface assaults and desiccation of underlying tissues through a tightly controlled program of regeneration and terminal differentiation which is significantly impacted by the activity of several members of the nuclear receptor (NR) superfamily. As such, there is significant interest in physiological and pharmacological control of select NRs. NRs are usually considered quintessential examples of constrained structure-function relationships among protein families because of amino acid identity and sequence subserving physical requirements inherent to a relatively centrally-located DNA-binding domain and carboxyl-terminal ligand-recognition domain which together lead to agonist-activated gene expression. Nevertheless, across the superfamily the amino terminus of many NR is an often-critical contributor in degree of receptor-dependent transcriptional activity despite little in apparent sequence similarity that might be instructive in understanding this ability. By looking beyond shared strict amino acid sequence identity, a number of investigations are revealing the “unstructured-function consequences of this disparity. Significant correlations between in silico and in vitro biophysical assessments are highlighting the shared trait of the unstructured nature or intrinsic disorder (ID) of NR amino termini and related functional consequences. Rather than the limited protein sequence variation-on-a-theme seen for zinc fingers (DNA binding) or a hydrophobic pocket (ligand binding), these amino-termini show sequence order diversity but often strikingly shared amino acid composition profiles not supporting a one-sequence–one-structure conformation. In this review, we look to integrate amino-termini ID reported in the literature, or predicted here, for select keratinocyte-expressed NR. As evidenced by success in drug targeting the amino-terminus of the androgen receptor, increased appreciation of amino-termini structure - or unstructure - might provide better understanding of NR function in general and possible future investigations on pharmacologic control over keratinocyte regeneration and/or differentiation.

Clinical development of CAR-T therapy for refractory multiple myeloma

A chimeric antigen receptor (CAR) comprises an extracellular ligand recognition domain linked to CD3ζ and induces T-cell activation upon antigen binding. Recently, the potential of CD19-targeted CAR T-cells (CAR-T) to treat multiple myeloma has been explored. A group in the University of Pennsylvania reported that 4 out of 10 patients with refractory myeloma achieved an objective response (sCR: 1, VGPR: 1, and PR: 2). Although the resultant cancer ablation was an arresting sight, it remains unclear whether CD19 is a suitable target for myeloma. Therefore, CAR-T therapy employing alternative target antigens is being attempted. Some current clinical trials are utilizing CAR against differentiation antigens, including CD138, CD38, and kappa light chain. B-cell maturation antigen (BCMA) is also a potential candidate. A group in the NCI described 12 patients with refractory myeloma enrolled in a BCMA-CAR phase 1 clinical trial. Two patients receiving the highest dosage attained an objective response. Nevertheless, considerable research is required for CAR immunotherapy to become universally available to patients with myeloma. The current enthusiasm for the development of this cutting-edge technology is justified because extraordinary evidence indicates that it is effective, although this may not apply to every case. CAR-T therapy for myeloma is currently undergoing rapid, wide-scale growth both in academia and in industry-sponsored clinical development.

Cell-Surface Expression Levels Are Important for Fine-Tuning the Performance of Receptor Tyrosine Kinase-Based Signalobodies.

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies. This study aims at fine-tuning the performance of the signalobodies based on three RTKs (fibroblast growth factor receptor 1, insulin receptor, and c-fms). To this end, the cell-surface expression levels of the RTK-based signalobodies were altered by locating their genes either upstream or downstream of the internal ribosomal entry site, and by inserting 1 to 3 alanine residue(s) at the intracellular juxtamembrane region. As a result, while the signaling response was different among the three signalobodies, the antigen-dependent on/off regulation became tighter when the cell-surface expression levels of the signalobodies were lowered. Therefore, we successfully developed a method to diminish the leaky signaling of RTK-based signalobodies, which will be important for establishing the signalobody-based platform technology that can produce cells of interest for regenerative medicine.

Abstract 4983: T cells expressing chimeric PD1 receptors that contain a Dap10 costimulatory domain are a potential treatment for multiple types of cancer

Abstract Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular ligand recognition domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, and colon cancer cell lines was measured. Of the ten cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Kelsey Deal, Emily Nylen, Amorette E. Barber. T cells expressing chimeric PD1 receptors that contain a Dap10 costimulatory domain are a potential treatment for multiple types of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2017-4983

Expression and protective role of two novel NACHT-containing proteins in pathogen infection

Lower vertebrates have been found to possess over 200 NACHT-domain encoding genes; but, to date, very little is known about their functional activity. This article describes the sequences and expression analysis of two zebrafish NACHT-containing proteins, namely NALPL1 and NALPL2. In addition, the functions of zebrafish NALPL1 and NALPL2, which are absent for both amino-terminal effector-binding domain (EBD) and carboxy-terminal ligand-recognition domain (LRD), were investigated for the first time in fish species. The predicted NALPL1 and NALPL2 proteins consist of 651 and 847 amino acids (aa), respectively, with both molecules only containing NACHT domain, which were different from other NACHT-family members. Phylogenetic analysis showed that zebrafish NALPL1 and NALPL2 have a closer relationship with mammalian NALP subfamily than NOD subfamily. The differential expression patterns of NALPL1 and NALPL2 in development stages and organs were observed, suggesting the difference of action phase and effector organ of NALPL1 and NALPL2. When the modulation of NALPL1 and NALPL2 in pathogen infection was analyzed, it was found that the two molecules were upregulated by both bacterial and viral infection. Overexpression of NALPL1 and NALPL2 resulted in significant inhibition for intracellular Edwardsiella tarda growth. Further studies demonstrated that NALPL1 and NALPL2 also contributed to protection against viral infection. These results demonstrate that both NALPL1 and NALPL2 are important intracellular proteins in host surveillance against both bacterial and viral infection. Interestingly, the expression of downstream signaling genes was not affected by the overexpression of NALPL1 or NALPL2, but NOD1 and MDA5 were upregulated by NALPL1 or NALPL2 overexpression, suggesting that they likely act in pathogen infection through the interaction with other PRRs.

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment

Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N- and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes

Aims: Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOXOriginal Research Article International Journal of Biochemistry Research & Review, 4(4): 284-294, 2014 285 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10 −7 M and that of the C70–peptide complex was 6.6 × 10 −8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.

Crystal Structure of the Sema-PSI Extracellular Domain of Human RON Receptor Tyrosine Kinase

Human RON (Recepteur d’Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors’ exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON splice variant, RONΔ85.

Structural Analysis of SHARPIN, a Subunit of a Large Multi-protein E3 Ubiquitin Ligase, Reveals a Novel Dimerization Function for the Pleckstrin Homology Superfold

SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.

Discovery and Development of α7 Nicotinic Acetylcholine Receptor Modulators

Efforts in the design and discovery of selective α7 nicotinic acetylcholine receptor (nAChR) agonists were both facilitated and hampered by observation that ligands can show dual affinity at both the 5-HT3 receptor (5-HT3R) and nAChR. 1 The 5-HT3R and nAChRs are both part of the Cys-loop superfamily of ligand-gated ion channels. Further, there is significant sequencehomology between 5-HT3R and α7nAChR inthe ligand recognition domain. 2 Previously reported potent α7 nAChR agonists lacked selectivity versus 5-HT3R, 3 and antagonist activity at 5-HT3R often translated into agonism at α7 nAChR. The crossover in affinity might be explained by pharmacophoric elements common to both 5-HT3R and α7 nAChR: a basic amine (protonated at physiological pH) provides for a cation� π interaction; a hydrogen-bond acceptor, e.g., a carbonyl group, forms a hydrogen bond; and aromatic moieties participate in π� π interactions. 4 In view of reported side effects, i.e., constipation, asymptomatic electrocardiogram changes, and arrhythmias, associated with 5-HT3R antagonists, 5 efforts of research groups were focused on design of ligands specifically interacting with only the α7 nAChR to maximize the therapeutic effect and minimize the adverse effects. Over the past 10 years, drug discovery efforts significantly expanded the quantity and quality of selective α7 nAChR ligands. Those have been summarized in several excellent reviews. 6� 8 Despite early skepticism centered around the rapid desensitization of the α7 nAChR and the hypothesis that agonists might not be functional agonists in vivo, 10 of them have already been advanced to clinical trials. In this review, we highlight the most advanced and characterized (especiallyinvivo)selective α7nAChRorthostericandallosteric α7 nAChR modulators. 1. AGONISTS

Induction of tolerance and immunity by redirected B cell-specific cytolytic T lymphocytes

Chimeric receptors bearing ligand recognition domains linked to signaling regions from the T-cell receptor can redirect T lymphocytes against non-MHC-restricted targets. Cytolytic T lymphocytes (CTL) expressing these chimeric receptors are being tested in preclinical and clinical trials for activity in cancer, infectious diseases and autoimmunity. The chimeric receptors may incorporate antigenic epitopes previously unrecognized by the immune system. Whether a receptor-specific antibody response develops to these neoantigens and whether such a response inhibits therapeutic cell activity is unknown. We hypothesized that upon engagement of a chimeric receptor-specific B cell, receptor-modified CTL will be activated, lysing the B cell and inducing tolerance to the chimeric receptor rather than immunity. We demonstrate that receptor-modified CTL are indeed stimulated by cognate receptor-specific B cells, proliferate and produce cytokines in response and kill the B cells in vitro and in vivo. However, this is insufficient to induce full B-cell tolerance. Modified CTL induce a chimeric receptor-specific antibody response independent of any other source of antigen. Nevertheless, the CTL retain substantial activity even in the presence of saturating doses of receptor-specific antibody. Thus antichimeric receptor antibody responses need to be considered in the clinical use of chimeric receptor-modified T cells. However, the inhibitory activity of these antibodies may in cases be limited.

Ligand-induced Structural Changes of the CD44 Hyaluronan-binding Domain Revealed by NMR

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits.

Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely, T cell receptor zetacyt, CD3epsiloncyt, CD3deltacyt, and CD3gammacyt, B cell receptor Igalphacyt and Igbetacyt, and Fc receptor FcepsilonRIgammacyt, using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that zetacyt, CD3epsiloncyt, and FcepsilonRIgammacyt bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of zetacyt and FcepsilonRIgammacyt does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.

Existence of Different but Overlapping IgG- and IgM-Binding Sites on the Globular Domain of Human C1q

C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined their interactions with IgM. The comparative analysis of IgM- and IgG-binding abilities of the mutants suggests that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap. Whereas Arg(B108), Arg (B109), and Tyr(B175) mainly constitute the IgM-binding site, the residues Arg(B114), Arg(B129), Arg(B163), and His(B117) that have been shown to be central to IgG binding are not important for the C1q-IgM interaction. Given the location of Arg(B108), Arg (B109), and Tyr(B175) in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain.

Interaction of C1q with IgG1, C-reactive Protein and Pentraxin 3: Mutational Studies Using Recombinant Globular Head Modules of Human C1q A, B, and C Chains

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.

Diagnostic cross‐linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg(E) from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg(E) domain was created using the homodimeric, four-helix bundle structure of the Tar(S) domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg(E) periplasmic domain is very similar to the Tar(S) domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.

The Molecular Functions of Nod Proteins and their Associated Diseases

Nod proteins are defined as proteins carrying nucleotide-oligomerization domains (NODs) and are involved in regulation of immune responses and apoptosis. The Nod protein family contains 23 human members including Nod1, Nod2, cryopyrin, Ipaf, Apaf-1 and CIITA, as well as thousands of plant proteins, which are involved in pathogen-specific defense responses. A Nod protein generally contains an amino-termina l domain for binding downstream effector molecules, a central NOD and a carboxyl-terminal ligand recognition domain (LRD). Nod1 and Nod2 are involved in host recognition of small molecules that are components of bacterial peptidoglycan and activate nuclear factor κB (NF-κB) in response to sensing these molecules. This NF-κB activation occurs in a RICK- and IKK-dependent manner. The core ligand structure for Nod2 is muramyl dipeptide, a structural motif common in all bacteria, whereas the ligand for Nod1 is a dipeptide designated as iE-DAP, a motif found in only certain subgroups of bacteria. These molecules and their derivatives mediate host innate responses against bacteria and also function as immunostimula tory adjuvants through induction of cytokine secretion and co-stimulatory molecule expression. Although the mechanism is unknown, genetic and functional defects of Nod proteins are associated with several inflammatory diseases and immunodeficiency. These include susceptibility for Crohn's disease and Blau syndrome (Nod2), three related inflammatory diseases (cryopyrin) and type II bare lymphocyte syndrome (CIITA). Functional analyses of mutant Nod proteins suggest a common molecular basis for these diseases.

NMR structure and peptide hormone binding site of the first extracellular domain of a type B1 G protein-coupled receptor.

The corticotropin-releasing factor (CRF) ligand family has diverse effects on the CNS, including the modulation of the stress response. The ligands' effects are mediated by binding to CRF G protein-coupled receptors. We have determined the 3D NMR structure of the N-terminal extracellular domain (ECD1) of the mouse CRF receptor 2beta, which is the major ligand recognition domain, and identified its ligand binding site by chemical-shift perturbation experiments. The fold is identified as a short consensus repeat (SCR), a common protein interaction module. Mutagenesis reveals the integrity of the hormone-binding site in the full-length receptor. This study proposes that the ECD1 captures the C-terminal segment of the ligand, whose N terminus then penetrates into the transmembrane region of the receptor to initiate signaling. Key residues of SCR in the ECD1 are conserved in the G protein-coupled receptor subfamily, suggesting the SCR fold in all of the ECD1s of this subfamily.

Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function

AbstractRecent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+T cells using either Kb-ζ or Kb-CD28-ζ receptors had similar functional activities, although the CD28-ζ receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-ζ chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance. (Blood. 2003;102:4320-4325)

Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins and wild-type and lectin-gene-transfected tumor cells as targets.

Multivalent glycoclusters have the potential to become pharmaceuticals by virtue of their target specificity toward clinically relevant sugar receptors. Their application can also provide fundamental insights into the impact of two spatial factors on binding, i.e., topologies of ligand (branching mode, cluster presentation) and carbohydrate recognition domains in lectins. Persubstituted macrocycles derived from nucleophilic substitution of iodide from heptakis 6-deoxy-6-iodo-beta-cyclodextrin by the unprotected sodium thiolate of 3-(3-thioacetyl propionamido)propyl glycosides (galactose, lactose and N-acetyllactosamine) were prepared. The produced glycoclusters were first tested as competitive inhibitors in solid-phase assays. A plant toxin from mistletoe and an immunoglobulin G fraction from human serum were markedly susceptible. A nearly 400-fold increase in inhibitory potency of each galactose moiety in the heptavalent form relative to free lactose (217-fold relative to free galactose) was detected. Thus, these glycoclusters can efficiently interfere, for example, with xenoantigen-dependent hyperacute rejection. Among the tested galectins selected from this family of adhesion- and growth-regulatory endogenous lectins, the substituted beta-cyclodextrins acted as sensors to delineate topological differences between the two dimeric prototype proteins. The relatively strong reactivity with chimera-type galectin-3, a mediator of tumor metastasis, disclosed selectivity for glycocluster binding among galectins. Equally important, the geometry of ligand display (maxiclusters, bi- or triantennary N-glycans) made its mark on the inhibitory potency. To further determine the sensitivity of a distinct galectin presented on the cell surface and not in solution, we established a stably transfected tumor cell clone. We detected a significant response to presence of the multivalent inhibitor. This type of chemical scaffold with favorable pharmacologic properties might thus be exploited for the design of galectin- and ligand-type-selective glycoclusters.