h erα h erβ lbd primary sequences  (GenScript corporation)

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Domain architecture of full-length ERs (NTD: N-terminal domain; AF1: activation-function 1; DBD: DNA-binding domain; LBD: ligand-binding domain; AF2: activation-function 2). b , Maximum-likelihood phylogenetic tree of 1,051 ER LBD sequences from vertebrates and protostomes; the branch corresponding to protostomes is compressed. c , X-ray crystal structure of the rf ERγ LBD solved at 1.95 Å resolution revealing conservation of protein folding, homodimer assembly, AF2 conformation and d E2 binding mode. Secondary structure elements are labelled in bold and residues are labelled accordingly (using h ERα LBD numbering for consistency). In d , residues that are conserved with ERα and ERβ homologs from human and rainbowfish are coloured in black. Sidechains and E2 are shown as sticks, and the simulated annealing composite omit 2F o -F c electron density map (1.5 σ) is shown as blue mesh. e , Representative snapshots of the F425 χ 1 sidechain dihedral angle obtained from K-means clustering (k=10) of triplicate 1 µs all-atom molecular dynamics (MD) simulations. Residue sidechains and E2 are shown as sticks (E2: yellow; F/M/I421: orange; F425: green; I/L429: purple). f , Histogram showing aggregate F425 χ 1 sidechain dihedral angle populations for each homolog. g , Top-down perspective of the rf ERγ LBD homodimer showing structural plasticity of the dimerisation interface; notable residues are shown as sticks and coloured according to the legend below. h , Hydrophobic surface representation of one subunit from the rf ERγ LBD homodimer and opposing residue sidechains.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Activation Assay, Binding Assay, Ligand Binding Assay, Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, An alignment comparing the primary amino acid sequences of each homolog. The numbering at sequence ends are the start and finish residue positions of each LBD; however, herein h ERα LBD numbering is used for consistency. Residue positions encoding the ligand binding pocket (LBP) are shown in green; the activation function-2 interface (AF2) in orange; and the dimerisation interface shown in purple. b, X-ray crystal structures of the h ERα LBD bound to estradiol (E2) and h SRC1 coactivator peptide (PDB: 3UUD); the rf ERα LBD bound to E2 and h SRC2 coactivator peptide (PDB: 9D8Q); the h ERβ LBD (PDB: 3OLL) bound to E2 and h SRC1 coactivator peptide; a computational homology model of the rf ERβ LBD bound to E2; and X-ray crystal structure of the rf ERγ LBD (PDB: 9D8R) bound to E2. Each structure is shown as the homodimer, with the second subunit coloured in grey. c, Close-up perspective of the LBP showing the conservation of E2 binding mode across homologs. d, The binding mode of coactivator peptides solved with h ERα, rf ERα and h ERβ, and all peptides modelled onto the AF2 of rf ERβ and rf ERγ LBDs, revealing conservation of structure and LxxLL motif recognition.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Sequencing, Residue, Ligand Binding Assay, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, Modelling of all possible rotamer orientations of the F421 substitution in ERα and ERβ backgrounds with the Dunbrack rotamer library shows clashing of the phenyl sidechain with E2 and neighbouring structure, compared to the wildtype amino acid. b, Comparison of the structure and sequence of H6 between homologs. c, Secondary structure analysis of H6 (residues 409 to 417) calculated from a 1 µs all-atom molecular dynamics (MD) simulation using the DSSP algorithm in MDTraj , .

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Comparison, Sequencing

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Schematic outlining the approach. See Materials and Methods for a detailed description and breakdown of each step. b , A contact matrix showing all non-covalent residue interactions between residues (all homologs) coloured by probability. c , Graph of the allosteric network comprised of components 1 and 2 (C1: purple; C2: blue). Node size is proportional to the betweenness centrality (or importance) of each node. d , Allosteric network mapped to the crystal structure of the wildtype h ERα LBD (PDB: 1GWR). Residue sidechains are shown as spheres and coloured by component. e , Structural analysis of the allosteric network. Conserved residues shared are labelled in black while non-conserved residues are labelled in red with varying amino acids annotated. f , Same as in c but for the components deemed important for supporting protein folding. g , The folding network mapped to structure. Residue sidechains are shown as spheres and coloured by component (see key). h ) Perspective of the α-helical bundle and folding network components. Residue annotation follows convention of d .

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: Left: backbone heavy-atom RMSD for each 1 µs replicate; middle: estradiol (E2) RMSD for each 1 µs replicate; right: helix-12 (H12) backbone RMSD for each 1 µs replicate. a-e, in order: h ERα LBD, h ERβ LBD, rf ERα LBD, rf ERβ LBD and rf ERγ LBD. The 5 ns rolling-average (solid line) is superimposed above raw RMSD values.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques:

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Evaluation of variability within functional regions of 1,051 ER LBD sequences (α, β and γ) from phylogenetically diverse taxa. Area under the curve (AUC) was used as a quantitative measure of constraint, calculated by plotting the fraction of unique genotypes in the alignment encoding functional regions (e.g., the dimerisation interface) against the fraction of the total sequence population. b , The evolutionary conservation of each residue position along the length of the LBD determined by the Jensen-Shannon divergence (JSD). c , Evolutionary conservation of each residue position encoding the ligand binding pocket (LBP), activation function-2 (AF2), allosteric network (AN), dimerisation interface (DI), folding network (FN) and non-functional residues (NF), shown as a box plot. The box plot shows the median and interquartile range (IQR); whiskers represent the distribution of the data as a function of the IQR. Mann-Whitney U-test was used to determine statistical significance compared to NF residues (LBP: P = 0.00095; AF2: P = 0.00303; AN: P = 0.03354; DI: P = 0.12424; FN: P = 0.10295). d , The evolutionary conservation of each residue position mapped to the h ERα LBD (1GWR; missing loops were modelled in ICM-Pro Molsoft for visualisation purposes) highlights the architecture of constraint within the ER LBD. Each residue position comprising regions are shown as spheres (Cα atom) and coloured according to the level of conservation (red = low; blue = high). Refer to for the network analysis.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Sequencing, Residue, Ligand Binding Assay, Activation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a-d, Sequence space networks of the ligand binding pocket (LBP), activation function-2 interface (AF2), allosteric network and dimerisation interface. A genotype is depicted as a node and are connected by an edge to another node if the Hamming distance between them is equal to 1, or 1 residue substitution (i.e., the Hamming distance between AGFST and AFFST is 1). The colour of each node corresponds to the number of times each genotype is observed in the alignment of 1,051 ER LBD sequences. For visual clarity only connected nodes are shown. e, The number of mutational steps that each genotype (a line) must take to visit all other genotypes in the network, given as the Hamming distance, or shortest-mutational paths. The solid orange line represents the mean trajectory.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Sequencing, Ligand Binding Assay, Activation Assay, Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a ) The burden, or total number, of unique variants within each functional region shown as a grouped barplot (LBP: ligand binding pocket; AF2: activation function-2; AN: allosteric network; DI: dimerisation interface; FN: folding network; NF: non-functional residues). Note that a residue position may harbour more than one unique variant. b , Venn diagram showing the distribution of shared residue positions harbouring variants between h ERα and h ERβ LBDs. c , Histogram showing the distribution of the evolutionary conservation (Jensen-Shannon divergence) of each variant residue position for both h ERα and h ERβ, as well as residue positions that share variant occurrence. d , Average solvent accessible surface area (SASA) of residue positions harbouring variants calculated from the homodimeric crystal structures of the h ERα (PDB: 1GWR) and h ERβ (PDB: 3OLL) LBD. Missing residues in loops were modelled with ICM-Pro Molsoft. e , Average Cα-atom root-mean squared fluctuations (RMSF) of residue positions harbouring variants calculated from the replicate MD simulations. f , Molecular architecture of residue positions harbouring variants mapped to h ERα (PDB: 1GWR) and h ERβ (PDB: 3OLL) LBDs. Residues are shown as spheres (Cα atoms) and coloured according to variant burden. The black box highlights the differences between variant burden in ligand binding pocket and of allosteric network residues between h ERα and h ERβ LBDs. Whiskers in d and e represent median and interquartile range (IQR). g , A structural perspective of the ligand binding pocket and allosteric network residues of the h ERα LBD (left) and h ERβ LBD (right). Important residue sidechains are shown as sticks. Residue positions with variants are identified by a sphere which is coloured to the total burden of unique variants. Wildtype residues are labelled in black, while identified variants are labelled in red to highlight changes. h , Structural comparison between wildtype h ERα (PDB: 1GWR) and Y537S (gain-of-function; PDB: 3UUD). Adjacent allosteric network residues are shown as sticks and the residue at position 537 is coloured teal

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Ligand Binding Assay, Activation Assay, Residue, Variant Assay, Solvent, Comparison

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, Burden of unique missense variants across each position of the h ERα and h ERβ LBD. Variants were obtained from gnomAD( 48 ). b, Significance p-values calculated from hypergeometric tests for statistical depletion of variants in each functional region relative to all positions within the LBD. A significance threshold of P = 0.05 was used. Only the ligand binding pocket (LBP) and allosteric network (AN) in the h ERα LBD were significantly depleted of variants, as compared to the activation function-2 interface (AF2), dimerisation interface (DI) and folding network (NF). No functional region was significantly depleted of variants in the h ERβ LBD. c, Structural modelling of example h ERα variants M421V (putative benign) and R394S, (putative loss-of-function) and Y537S (gain-of-function; PDB: 3UUD).

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Ligand Binding Assay, Activation Assay