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1.
Figure 3

Figure 3. Three historical substitutions in AncCR recapitulate the evolution of reduced sensitivity.. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

Shown is the log EC50 of molar DOC with the highly sensitive ancestor (AncCR, gray), the low sensitivity descendent (AncGR1.1, yellow), and AncCR mutants. Single and combination mutants are denoted with colored asterisks: V43A, pink; R116H, blue; C71S, orange.

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.
2.
Figure 5

Figure 5. The structural context of site 71.. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

S71 (orange) in AncGR1.1 (Chain B, yellow) forms a water-mediated hydrogen bond with the carbonyl carbon of L139 and the T143 side chain. In AncCR, the side chain of an engineered S71 mutant adopts a slightly different conformation (inset). The C71S substitution increases the hydrophilicity of the region. Also shown are the relative locations of the ligand, DOC (green); V43A (pink); the R116H and Q113K substitutions (blue); possible hydrogen bonding (red dashes); and relevant helices (light grey text).

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.
3.
Figure 2

Figure 2. AncGR1 and its descendents evolved reduced hormone sensitivity.. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

Ligand-dependent transcriptional activation of receptors was measured in the presence of increasing concentrations of hormone using a luciferase reporter gene assay. Dose-response curves were calculated for receptor-hormone pairs and plotted as the log effective concentration of hormone for half maximal activation (log EC50) in molar with standard error. Larger values, lower sensitivity; smaller values, higher sensitivity. Hormones are: 11-deoxycorticosterone (DOC), corticosterone (B), 11-dehydrocorticosterone (11-DHC), and 1α-hydroxycorticosterone (1α-B). Cartilaginous fish species are shown in (Figure 1). 11-DHC did not activate cartilaginous fish GRs in our assay (defined as <2-fold activation for EC50>1 µM of hormone) and is not shown for these receptors.

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.
4.
Figure 6

Figure 6. The effect of historical GR substitutions on AncCR protein stability, predicted by FoldX [43].. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

A) The distribution of stability effects (ΔΔG) for almost all (n = 29 of 36) single GR substitutions in the AncCR background. Colored asterisks indicate bins with notable mutants: V43A (pink), R116H (blue), and C71S (orange). B) For those mutants in which hormone sensitivity was assessed (n = 10, plus AncCR), the predicted loss of stability correlates well with the observed loss of sensitivity towards DOC. Linear regression with C71S mutants, r2 = 0.78; without, r2 = 0.89. Single and combination mutants are colored as in (A). X values are the average of five FoldX runs with standard deviation; Y values are the mean of triplicate reactions with standard error. The intercept lies at values for AncCR (0, −10.870).

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.
5.
Figure 4

Figure 4. The crystal structure of AncGR1.1-LBD in complex with DOC (yellow, PDB 3RY9) compared to the previously solved structure of AncCR with DOC (gray, PDB 2Q3Y).. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

Green sticks indicate DOC; possible hydrogen bonding indicated by red dashes. Helices are indicated by light grey text; AF-H equals H12, the activation function helix. A) AncCR and AncGR1.1 are highly structurally conserved despite 36 substitutions between them. Spheres show sequence differences: pink, position 43; blue, position 116; orange, position 71. B) and C) show the structural environment around position 43. The substitution of V43 in AncCR (B) in place of A43 in AncGR1.1 (C) is thought to weaken the hydrophobic interactions of the surrounding helices. D) and E) show the structural neighborhood of position 116. Replacing R116 (D) with H116 (E), along with a Q113K substitution, significantly reduces and rearranges ancestral hydrogen bonding.

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.
6.
Figure 1

Figure 1. Simplified phylogeny of corticosteroid receptors.. From: Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor.

Ancestral sequences are shown at relevant nodes: AncCR, the last common ancestor of all MRs and GRs; AncGR1, the GR ancestor of cartilaginous fishes and bony vertebrates; AncGR2, the GR ancestor of ray- and lobe-finned fishes (including tetrapods); AncMR1, the MR ancestor of cartilaginous fishes and bony vertebrates. (AncGR1.0 and AncGR1.1 are different reconstructions of node AncGR1, inferred from datasets with different taxon sampling.) Black, high sensitivity receptors; gray, low sensitivity receptors. Single and double gray dashes mark functional shifts towards reduced sensitivity and increased specificity, respectively. Support values are the chi-square statistic (1 – p, where p equals the estimated probability that a node could occur by chance alone) calculated from approximate likelihood ratios. The length of branches from AncCR to AncMR1 and to AncGR1, expressed as the mean number of substitutions per site, are indicated in parentheses.

Sean Michael Carroll, et al. PLoS Genet. 2011 June;7(6):e1002117.

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