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Evolution of a New Function by Degenerative Mutation in Cephalochordate Steroid Receptors

Figure 7

Mechanism for evolution of a duplicate receptor repressor.

A) Conservation and variability of ligand-contacting residues. The figure shows a generic steroid hormone surrounded by the amino acids that line the ligand-binding pocket in the crystal structures of the human ERα. For each site, residues from the human ERα (HsER), BfSR, BfER, and AncSR1 are shown, from top to bottom, numbered according to the human ERα sequence. Moieties at the 3 and 17 positions, shown as large circles, vary among steroids. B) Historical substitutions R394C and F404L, which occurred in the lineage leading to transcriptional repressor BfER, are predicted to abolish estrogen binding and activation. Left, x-ray crystal structure of the ligand pocket of the human ERα with estradiol. Arg394 and Phe404 play key roles in a network of hydrogen bonds and packing interactions that stabilize the ligand and transcriptionally active conformation. Right, mutations R394C and F404L from BfER disrupt this network. Red sphere, water molecule. C) Mutations R394C and F404L, introduced into BfSR, abolish the receptor's transcriptional capacity and generate a dose-dependent repressor of the wild-type (WT) BfSR. Numbers show the quantity of each plasmid transfected (in ng) with 1 µM estradiol (black bars) or with no hormone added (vehicle only, white bars). Mutation C205A, which disrupts DNA-binding, eliminates BfER's capacity to repress BfSR-driven transcription. D) Either mutation R394C or F404L, introduced singly into BfSR, are each sufficient to abolish transcriptional capacity and generate a repressor of BfSR-WT.

Figure 7