Five amino acid residues in cysteine-rich domain of human T1R3 were involved in the response for sweet-tasting protein,thaumatin

Thaumatin, a sweet-tasting plant protein, elicits a sweet taste sensation at 50 nM in humans but not rodents. Although it was shown that the cysteine-rich domain (CRD) of human T1R3 (hT1R3) is important for the response to thaumatin, the amino acid residues within CRD critical for response are still unknown. A comparison of the amino acid sequence (69 amino acid residues) of CRD between hT1R3 and mouse T1R3 (mT1R3) revealed sixteen amino acids that differ.     

Solution structure of the chick TGFbeta type II receptor ligand-binding domain

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.     

Insights into function of PSI domains from structure of the Met receptor PSI domain

PSI domains are cysteine-rich modules found in extracellular fragments of hundreds of signaling proteins, including plexins, semaphorins, integrins, and attractins. Here, we report the solution structure of the PSI domain from the human Met receptor, a receptor tyrosine kinase critical for proliferation, motility, and differentiation. The structure represents a cysteine knot with short regions of secondary structure including a three-stranded antiparallel beta-sheet and two alpha-helices. All eight cysteines are involved in disulfide bonds with the pattern consistent with that for the PSI domain from Sema4D. Comparison with the Sema4D structure identifies a structurally conserved core comprising the N-terminal half of the PSI domain. Interestingly, this part links adjacent SEMA and immunoglobulin domains in the Sema4D structure, suggesting that the PSI domain serves as a wedge between propeller and immunoglobulin domains and is responsible for the correct positioning of the ligand-binding site of the receptor.     

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.     

Gene conversion and the evolution of three leucine-rich repeat gene families in Arabidopsis thaliana

The high number of duplicated genes in plant genomes provides a potential template for gene conversion and unequal crossing-over. Within a gene family these two processes can render all members homogeneous or generate diversity by reassorting variants among paralogs. The latter is especially feasible in families where gene diversity confers a selective advantage and thus conversion events are likely to be retained. Consequently, the most complete record of gene conversion is expected to be most evident in gene families commonly subjected to positive selection. Here, we describe the extent and characteristics of gene conversion and unequal crossing-over in the coding and noncoding regions of nucleotide-binding site leucine-rich repeat (NBS-LRR), receptor-like kinases (RLK), and receptor-like proteins (RLP) in the plant Arabidopsis thaliana. Members of these three gene families are associated with disease resistance and their pathogen-recognition domain is a documented target of positive selection. Our bioinformatic approach to study the major family features that may influence gene conversion revealed that in these families there is a significant association between the occurrence of gene conversion and high levels of sequence similarity, close physical clustering, gene orientation, and recombination rate. We discuss these results in the context of the overlap between gene conversion and positive selection during the evolutionary expansion of the NBS-LRR, RLK, and RLP gene families.     

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.     

Local motifs involved in the canonical structure of the ligand-binding domain in the nuclear receptor superfamily

Structural and sequence alignment analyses have revealed the existence of class-dependent and -independent local motifs involved in the overall fold of the ligand-binding domain (LBD) in the nuclear receptor (NR) superfamily. Of these local motifs, three local motifs, i.e., AF-2 fixed motifs, were involved in the agonist conformation of the activation function-2 (AF-2) region of the LBD. Receptor–agonist interactions increased the stability of these AF-2 fixed motifs in the agonist conformation. In contrast, perturbation of the AF-2 fixed motifs by a ligand or another protein molecule led the AF-2 architecture to adopt an antagonist conformation. Knowledge of this process should provide us with novel insights into the ‘agonism’ and ‘antagonism’ of NRs.     

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.     

Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6 R) was constructed by computer-guided homology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hIL-6R with the ligand (hIL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hIL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cys121, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hIL-6 R and hIL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hIL-6R and hIL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hIL-6R with its ligand, and the active pocket may be used as a target for designing small hIL-6R-inhibiting molecules in our further study.     

Therapeutic effects of rosuvastatin in hypercholesterolemic prediabetic mice in the absence of low density lipoprotein receptor

Statins are effective drugs used to prevent and treat cardiovascular diseases but their effects in the absence of low density lipoprotein receptor (LDLR) and on the risk of diabetes are not yet well characterized. The aim of this study was to clarify systemic and pleiotropic effects of rosuvastatin on cardiovascular and diabetic phenotypes.IGF-II/LDLR^?/?ApoB^100/100 hypercholesterolemic prediabetic mice were used to test the effects of rosuvastatin on plasma glucose, insulin, lipids, atherosclerosis and liver steatosis. To get a more comprehensive view about changes in gene expression RNA-sequencing was done from the liver.Rosuvastatin significantly reduced plasma cholesterol in hypercholesterolemic mice in the absence of LDLR but had no effects on atherosclerosis at aortic sinus level or in coronary arteries. Rosuvastatin also significantly reduced liver steatosis without any harmful effects on glucose or insulin metabolism. RNA-sequencing showed relatively specific effects of rosuvastatin on genes involved in cholesterol metabolism together with a significant anti-inflammatory gene expression profile in the liver. In addition, significant changes were found in the expression of Perilipin 4 and 5 which are involved in lipid droplet formation in the liver. For the first time it could be shown that Tribbles proteins are affected by rosuvastatin treatment in the hyperlipidemic mice. Rosuvastatin had several positive effects on hypercholesterolemic mice showing early signs of diabetes, many of which are unrelated to cholesterol and lipoprotein metabolism. These results increase our understanding about the systemic and pleiotropic effects of rosuvastatin in the absence of LDLR expression.     

Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hlL-6R with the ligand (hlL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hlL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cyst 21, Cys132, Cys165, Cys1     

Solution structure and functional analysis of the cysteine-rich C1 domain of kinase suppressor of Ras (KSR).

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to promote signal transmission from Raf-1 to MEK and MAPK. All KSR proteins contain a conserved cysteine-rich C1 domain, and studies have implicated this domain in the regulation of KSR1 subcellular localization and function. To further elucidate the biological role of the KSR1 C1 domain, we have determined its three-dimensional solution structure using nuclear magnetic resonance (NMR). We find that while the overall topology of the KSR1 C1 domain is similar to the C1 domains of Raf-1 and PKCgamma, the predicted ligand-binding region and the surface charge distribution are unique. Moreover, by generating chimeric proteins in which these domains have been swapped, we find that the C1 domains of Raf-1, PKCgamma, and KSR1 are not functionally interchangeable. The KSR1 C1 domain does not bind with high affinity or respond biologically to phorbol esters or ceramide, and it does not interact directly with Ras, indicating that the putative ligand(s) for the KSR1 C1 domain are distinct from those that interact with PKCgamma and Raf-1. In addition, our analysis of the chimeric proteins supports the model that Raf-1 is a ceramide-activated kinase and that its C1 domain is involved in the ceramide-mediated response. Finally, our findings demonstrate an absolute requirement of the KSR1 C1 domain in mediating the membrane localization of KSR1, a crucial feature of its scaffolding activity. Together, these results underscore the functional specificity of these important regulatory domains and demonstrate that the structural features of the C1 domains can provide valuable insight into their ligand-binding properties.     

Crystal structure of YHI9, the yeast member of the phenazine biosynthesis PhzF enzyme superfamily

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.     

Novel cysteine-rich secretory protein in the buccal gland secretion of the parasitic lamprey, Lethenteron japonicum

Lampreys are one of the most primitive vertebrates diverged some 500 million years ago. It has long been known that parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We found two major protein components of 160 and 26 kDa in the buccal gland secretion of parasitic river lamprey, Lethenteron japonicum. The larger protein was identified as river lamprey plasma albumin. The complete primary structure of the 26-kDa protein was determined by protein and cDNA analysis. It belonged to the cysteine-rich secretory protein (CRISP) superfamily that includes recently identified reptile venom ion-channel blockers. Lamprey CRISP blocked depolarization-induced contraction of rat-tail arterial smooth muscle, but showed no effect on caffeine-induced contraction. The result suggests that lamprey CRISP is an L-type Ca(2+)-channel blocker and may act as a vasodilator, which facilitates the parasite to feed on the host's blood. The lamprey CRISP protein contains a number of short insertions throughout the sequence, when aligned with reptilian venom CRISP proteins, probably due to the large evolutionary distance between the Agnatha and the Reptilia, and may represent a novel class of venom CRISP family proteins.