Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.     

Enhanced auditory brainstem response and parental bonding style in children with gastrointestinal symptoms

Background

The electrophysiological properties of the brain and influence of parental bonding in childhood irritable bowel syndrome (IBS) are unclear. We hypothesized that children with chronic gastrointestinal (GI) symptoms like IBS may show exaggerated brainstem auditory evoked potential (BAEP) responses and receive more inadequate parental bonding.

Methodology/Principal Findings

Children aged seven and their mothers (141 pairs) participated. BAEP was measured by summation of 1,000 waves of the electroencephalogram triggered by 75 dB click sounds. The mothers completed their Children''s Somatization Inventory (CSI) and Parental Bonding Instrument (PBI). CSI results revealed 66 (42%) children without GI symptoms (controls) and 75 (58%) children with one or more GI symptoms (GI group). The III wave in the GI group (median 4.10 interquartile range [3.95–4.24] ms right, 4.04 [3.90–4.18] ms left) had a significantly shorter peak latency than controls (4.18 [4.06–4.34] ms right, p = 0.032, 4.13 [4.02–4.24] ms left, p = 0.018). The female GI group showed a significantly shorter peak latency of the III wave (4.00 [3.90–4.18] ms) than controls (4.18 [3.97–4.31] ms, p = 0.034) in the right side. BAEP in the male GI group did not significantly differ from that in controls. GI scores showed a significant correlation with the peak latency of the III wave in the left side (rho = −0.192, p = 0.025). The maternal care PBI scores in the GI group (29 [26]–[33]) were significantly lower than controls (31 [28.5–33], p = 0.010), while the maternal over-protection PBI scores were significantly higher in the GI group (16 [12]–[17]) than controls (13 [10.5–16], p = 0.024). Multiple regression analysis in females also supported these findings.

Conclusions

It is suggested that children with chronic GI symptoms have exaggerated brainstem responses to environmental stimuli and inadequate parental behaviors aggravate these symptoms.     

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.     

Tetrameric interaction of the ectoenzyme CD38 on the cell surface enables its catalytic and raft-association activities

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.     

Mutation of the plastidial alpha-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm

Plastidial phosphorylase (Pho1) accounts for approximately 96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30 degrees C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20 degrees C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.     

Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.     

Biochemical evidence for the heptameric complex L10(L12)6 in the Thermus thermophilus ribosome: in vitro analysis of its molecular assembly and functional properties

The stalk protein L12 is the only multiple component in 50S ribosomal subunit. In Escherichia coli, two L12 dimers bind to the C-terminal domain of L10 to form a pentameric complex, L10[(L12)(2)](2), while the recent X-ray crystallographic study and tandem MS analyses revealed the presence of a heptameric complex, L10[(L12)(2)](3), in some thermophilic bacteria. We here characterized the complex of Thermus thermophilus (Tt-) L10 and Tt-L12 stalk proteins by biochemical approaches using C-terminally truncated variants of Tt-L10. The C-terminal 44-residues removal (Delta44) resulted in complete loss of interactions with Tt-L12. Quantitative analysis of Tt-L12 assembled onto E. coli 50S core particles, together with Tt-L10 variants, indicated that the wild-type, Delta13 and Delta23 variants bound three, two and one Tt-L12 dimers, respectively. The hybrid ribosomes that contained the T. thermophilus proteins were highly accessible to E. coli elongation factors. The progressive removal of Tt-L12 dimers caused a stepwise reduction of ribosomal activities, which suggested that each individual stalk dimer contributed to ribosomal function. Interestingly, the hybrid ribosomes showed higher EF-G-dependent GTPase activity than E. coli ribosomes, even when two or one Tt-L12 dimer. This result seems to be due to a structural characteristic of Tt-L12 dimer.