Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptor

AbstractSequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix–helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2–H4 in the β2-adrenergic receptor (β2-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly3157.42 and Ser3197.46, on H7 for structure-function analysis. Replacing Ser3197.46 with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15–20% agonist-independent activity. Replacement of Ser3197.46 with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly3157.42 and Ser3197.46 are stabilizing β2-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly3157.42 with Trp2866.48 and hydrogen bonding interactions of Ser3197.46 with amino acids on H1–H2–H7 and with structural water

Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions

Diversity of structures and properties among catalases

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.Preparation of this manuscript was supported by Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada to P. C. LoewenPeer Reviewe

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase

<div><p>Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151–164 in Rac1 is essential for calmodulin binding. Within the 151–164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.</p> </div

An electrical potential in the access channel of catalases enhances catalysis

Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30-50 Å in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 Å from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 Å from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 Å distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues.This work was supported by Grant BIO2002-04419 from Direccion General de Investigacion Ciencia y Technologia (to I. F.) and Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P. C. L.)Peer Reviewe

Advanced Glycation End-Products Can Activate or Block Bitter Taste Receptors

Bitter taste receptors (T2Rs) are expressed in several tissues of the body and are involved in a variety of roles apart from bitter taste perception. Advanced glycation end-products (AGEs) are produced by glycation of amino acids in proteins. There are varying sources of AGEs, including dietary food products, as well as endogenous reactions within our body. Whether these AGEs are T2R ligands remains to be characterized. In this study, we selected two AGEs, namely, glyoxal-derived lysine dimer (GOLD) and carboxymethyllysine (CML), based on their predicted interaction with the well-studied T2R4, and its physiochemical properties. Results showed predicted binding affinities (Kd) for GOLD and CML towards T2R4 in the nM and &mu;M range, respectively. Calcium mobilization assays showed that GOLD inhibited quinine activation of T2R4 with IC50 10.52 &plusmn; 4.7 &mu;M, whilst CML was less effective with IC50 32.62 &plusmn; 9.5 &mu;M. To characterize whether this antagonism was specific to quinine activated T2R4 or applicable to other T2Rs, we selected T2R14 and T2R20, which are expressed at significant levels in different human tissues. A similar effect of GOLD was observed with T2R14; and in contrast, GOLD and CML activated T2R20 with an EC50 of 79.35 &plusmn; 29.16 &mu;M and 65.31 &plusmn; 17.79 &mu;M, respectively. In this study, we identified AGEs as novel T2R ligands that caused either activation or inhibition of different T2Rs

CaM is required for thrombin-induced activation of Rac1 in CHRF-288-11 cells and EGF-induced activation of Rac1 in HeLa cells.

<p>(A) CHRF-288-11 cells or (B) HeLa cells were serum starved for 12 h and incubated with W7 (150 μM) for 10 min followed by addition of thrombin to CHRF-288-11 cells and EGF to HeLa cells for 1 min or 3 min. At the end of the incubation cells were lysed using RIPA buffer. After centrifugation, 60 µl of the supernatant was suspended in 20 µl 4X Laemmli's sample buffer to determine level of endogenous Rac1 in various samples by western blotting. The rest of the supernatant was incubated with GST-PAK1 for 2 h at 4°C. After incubation, the beads were washed three times with Rac1 washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-Rac1 antibody. Quantification (adjusted for endogenous level of Rac1) was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding thrombin or EGF treatment. The experiments were repeated a minimum of three times. In part (C), an equal amount of lysate (500 µg) from HeLa cells transiently expressing wild type HA-Rac1 and stimulated for various times with EGF was incubated for 2 hrs at 4°C with anti-HA antibody coupled to agarose beads. At the end of incubation beads were washed and bound proteins analyzed using SDS-PAGE and western blotting using anti-CaM antibody. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different when compared to EGF at 0 min.</p

Effect of deletion of Rac1 putative CaM binding domain on interaction with purified bovine brain CaM.

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and GST-Rac1 mutant (amino acids 151 to 164 deleted) were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were WT GST-Rac1 or GST-Rac1 mutant beads with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. A representative autoradiograph and quantitation is shown above. The experiment was repeated a minimum of three times.</p

Activation of HA-Rac1 mutants is induced by EGF in HeLa cells.

<p>(A) Various HA-Rac1 mutants were transiently expressed in HeLa cells. 48 h post transfection cells were serum starved for 12 h and stimulated for 3 min with EGF and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, the supernatant was incubated with GST-PAK1 for 2 h at 4°C and the beads were washed three times with washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. Data presented is a representative immunoblot of at least three independent experiments. Quantification was carried out using Bio-Rad “quantity one” program and key * p<0.05 were considered significantly different. (B) GTP loading of WT Rac1 and mutants of Rac1 was tested by the GST-PAK1 pull-down assay. HeLa cells expressing various forms of HA-Rac1 were lysed in buffer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042975#s2" target="_blank">Materials & Methods</a>. After centrifugation, guanine nucleotides (100 µM GTPγS or 100 µM GDPβS) plus 10 mM EGTA (final concn.) were added to the supernatant and the mixture was incubated at 30°C for 15 min. At the end of the incubation, magnesium chloride (MgCl₂) was added to a final concentration of 60 mM to lock in nucleotides. The mixture was incubated with 100 µl GST-PAK1 beads for 2 h at 4°C. Unbound proteins were removed by washing three times with binding buffer. 30 µl of Laemmli's sample buffer was added to beads and heated at 100°C for 5 min. Western blotting was performed using mouse anti-HA antibody. In the figure above double refers to the Rac1 (K153A/R163A) mutant.</p