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Recognition Domain (recognition + domain)
Kinds of Recognition Domain Selected AbstractsA Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1HELVETICA CHIMICA ACTA, Issue 2 2007Romain Siegrist Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source] The lectin-complement pathway , its role in innate immunity and evolutionIMMUNOLOGICAL REVIEWS, Issue 1 2004Teizo Fujita Summary:, Innate immunity was formerly thought to be a non-specific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self. Recognition of pathogens is mediated by a set of pattern recognition receptors, which recognize conserved pathogen-associated molecular patterns (PAMPs) shared by broad classes of microorganisms, thereby successfully defending invertebrates and vertebrates against infection. Lectins, carbohydrate-binding proteins, play an important role in innate immunity by recognizing a wide range of pathogens. Mannose-binding lectin (MBL) and ficolin are lectins composed of a lectin domain attached to collagenous region. However, they use a different lectin domain: a carbohydrate recognition domain (CRD) is responsible for MBL and a fibrinogen-like domain for ficolin. These two collagenous lectins are pattern recognition receptors, and upon recognition of the infectious agent, they trigger the activation of the lectin-complement pathway through attached serine proteases, MBL-associated serine proteases (MASPs). A similar lectin-based complement system, consisting of the lectin,protease complex and C3, is present in ascidians, our closest invertebrate relatives, and functions in an opsonic manner. We isolated several lectins homologous to MBLs and ficolins and several MASPs in invertebrates and lower vertebrates, and herein we discuss the molecular evolution of these molecules. Based on these findings, it seems likely that the complement system played a pivotal role in innate immunity before the evolution of an acquired immune system in jawed vertebrates. [source] Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activityJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2005Jianya Y Huan Abstract Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the ,-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the ,empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. Copyright © 2004 Society of Chemical Industry [source] Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repairMOLECULAR MICROBIOLOGY, Issue 6 2008Samir Acharya Summary MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo. Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains. [source] Roles of the two ClpC ATP binding sites in the regulation of competence and the stress responseMOLECULAR MICROBIOLOGY, Issue 3 2001Kürsad Turgay MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10-fold but was not eliminated. In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC. [source] A novel missense mutation causing abnormal LMAN1 in a Japanese patient with combined deficiency of factor V and factor VIII,AMERICAN JOURNAL OF HEMATOLOGY, Issue 11 2009Takayuki Yamada Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is an inherited bleeding disorder characterized by a reduction in plasma concentrations of FV and FVIII. F5F8D is genetically linked to mutations in either LMAN1 or MCFD2. Here, we investigated the molecular basis of F5F8D in a Japanese patient, and identified a novel missense mutation (p.Trp67Ser, c.200G>C) in the LMAN1, but no mutation in the MCFD2. The amount of LMAN1 in Epstein-Barr virus-immortalized lymphoblasts from the patient was found to be almost the same as that in cells from a normal individual. Interestingly, an anti-MCFD2 antibody did not co-immunoprecipitate the mutant LMAN1 with MCFD2 in lymphoblasts from the patient, suggesting the affinity of MCFD2 for the mutant LMAN1 is weak or abolished by the binding of the anti-MCFD2 antibody. In addition, a Myc/6×His-tagged recombinant form of wild-type LMAN1 could bind to D-mannose, but that of the mutant could not. The p.Trp67Ser mutation was located in the carbohydrate recognition domain (CRD), which is thought to participate in the selective binding of LMAN1 to the D-mannose of glycoproteins as well as the EF-motif of MCFD2. Taken together, it was suggested that the p.Trp67Ser mutation might affect the molecular chaperone function of LMAN1, impairing affinity for D-mannose as well as for MCFD2, which may be responsible for F5F8D in the patient. This is the first report of F5F8D caused by a qualitative defect of LMAN1 due to a missense mutation in LMAN1. Am. J. Hematol. 2009. © 2009 Wiley-Liss, Inc. [source] Insecticidal action of mammalian galectin-1 against diamondback moth (Plutella xylostella)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 8 2009Shiang Jiuun Chen Abstract BACKGROUND: Previous studies showed that mammalian galectin-1 (GAL1) could interact with chitosan or chitin, one component of the peritrophic membrane (PM). This finding suggests that the PM could be a target of GAL1, which prompted the authors to explore the effect of GAL1 on larval growth and its potential mechanism. RESULTS: The development of Plutella xylostella (L.) larvae was significantly disturbed after they were fed recombinant GAL1. The histochemical structure and immunostaining pattern suggested that GAL1 treatment resulted in dose- and time-dependent disruption of the microvilli and abnormalities in these epithelial cells. Ultrastructural studies showed that the PM was not present in the midgut of GAL1-treated insects; instead, numerous bacteria were found in the lumen area. These results indicate that the protective function of the PM was disrupted by GAL1 treatment. Moreover, in vitro data showed that GAL1 interacts with chitosan/chitin in a dose-dependent manner, and also specifically binds to the PM in vitro. CONCLUSION: In view of the fact that the carbohydrate recognition domain of GAL1 recognises the structural motif N -acetyl lactosamine (Gal , 1,4 GlcNAc), which is similar to that of chitin (,-1,4 N -acetyl- D -glucosamine), it is proposed that the insecticidal mechanism of GAL1 involves direct binding with chitin to interfere with the structure of the PM. Copyright © 2009 Society of Chemical Industry [source] Enhancement of bound-state residual dipolar couplings: Conformational analysis of lactose bound to Galectin-3PROTEIN SCIENCE, Issue 7 2006Tiandi Zhuang Abstract Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein. [source] Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactoseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2008Veronika Krej, íková Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P4212 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10,Ĺ and preliminary X-ray diffraction data were collected to 3.2,Ĺ resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1,Ĺ. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4. [source] HCV RNA-dependent RNA polymerase replicates in vitro the 3, terminal region of the minus-strand viral RNA more efficiently than the 3, terminal region of the plus RNAFEBS JOURNAL, Issue 22 2001Sandrine Reigadas The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3, end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3, UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3, end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3, end of the minus-strand RNA: (a) the presence of a C residue as the 3, terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3, end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B. [source] Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repairMOLECULAR MICROBIOLOGY, Issue 6 2008Samir Acharya Summary MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo. Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains. [source] How C-type lectins detect pathogensCELLULAR MICROBIOLOGY, Issue 4 2005Alessandra Cambi Summary Glycosylation of proteins has proven extremely important in a variety of cellular processes, including enzyme trafficking, tissue homing and immune functions. In the past decade, increasing interest in carbohydrate-mediated mechanisms has led to the identification of novel carbohydrate-recognizing receptors expressed on cells of the immune system. These non-enzymatic lectins contain one or more carbohydrate recognition domains (CRDs) that determine their specificity. In addition to their cell adhesion functions, lectins now also appear to play a major role in pathogen recognition. Depending on their structure and mode of action, lectins are subdivided in several groups. In this review, we focus on the calcium (Ca2+)-dependent lectin group, known as C-type lectins, with the dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) as a prototype type II C-type lectin organized in microdomains, and their role as pathogen recognition receptors in sensing microbes. Moreover, the cross-talk of C-type lectins with other receptors, such as Toll-like receptors, will be discussed, highlighting the emerging model that microbial recognition is based on a complex network of interacting receptors. [source] | ||||||||||||||||