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Carboxyl-terminal Domain (carboxyl-terminal + domain)
Selected AbstractsMolecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domainFEBS JOURNAL, Issue 18 2002Yoko Kobayashi Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations , a closed, inactive form and an open, active form , differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure,function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a ,head-to-tail' orientation, two inactive LPL mutants , a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) , were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other. [source] Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirocheteFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2007Monica E. Embers Abstract Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection. [source] Mechanism of H-8 inhibition of Cyclin-dependent kinase 9: study using inhibitor-immobilized matricesGENES TO CELLS, Issue 3 2003Daisuke Shima Background: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-,-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. Results: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. Conclusions: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data. [source] Haplotype analysis of the human ,2-HS glycoprotein (fetuin) geneANNALS OF HUMAN GENETICS, Issue 1 2001M. OSAWA Alpha2-HS glycoprotein (AHSG), which is equivalent to fetuin in other species, is a protein found in human plasma. AHSG is polymorphic with two common alleles and many variants. To examine the intragenic haplotypes and their diversity at this locus, a contiguous genomic DNA sequence (10·3 kb) was analyzed in 20 samples (40 chromosomes), and haplotypes were determined for 309 subjects. Judging from the aligned nucleotide sequences and the conserved amino acid residues comparing human and chimpanzee AHSG, it was concluded that the type 1 allele is probably older and has evolved into four major suballeles. The type 2 allele was generated from one branch of the type 1 allele. AHSG*3 and *5 variants were each found to have a single nucleotide change in exon 7, resulting in the change of an amino acid residue from Arg299 to Cys and from Asp258 to Asn, respectively. It was noted that the AHSG*3 mutation gives rise to an additional cysteine residue, which possibly affects the conformation of the protein. The AHSG gene was found to have a low mutation rate and no apparent recombination events. Furthermore, the detected substitutions were nonhomogeneously distributed at this locus. In particular, four nonsynonymous substitutions were concentrated in the carboxyl-terminal domain. [source] Expression, purification, crystallization and preliminary crystallographic study of the carboxyl-terminal domain of the human voltage-gated proton channel Hv1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Shu Jie Li The voltage-gated proton channel Hv1 is essential to proton permeation and contains a voltage-sensor domain without a pore domain. It contains three predicted domains: an N-terminal acid and proline-rich domain, a transmembrane voltage-sensor domain and a C-terminal domain that is responsible for the dimeric architecture of Hv1. Here, the C-terminal domain of the human voltage-gated proton channel Hv1 (C-Hv1) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals have a tetragonal form and diffraction data were collected to 2.5,Å resolution in-house. The crystal belongs to space group P41212, with unit-cell parameters a = b = 37.76, c = 137.52,Å. Structural determination of C-Hv1 is in progress. [source] | |||||||||||||