A PROSS-designed extensively mutated estrogen receptor α variant displays enhanced thermal stability while retaining native allosteric regulation and structure

Protein stability limitations often hamper the exploration of proteins as drug targets. Here, we show that the application of PROSS server algorithms to the ligand-binding domain of human estrogen receptor alpha (hER alpha) enabled the development of variant ERPRS* that comprises 24 amino acid substitutions and exhibits multiple improved characteristics. The protein displays enhanced production rates in E. coli, crystallizes readily and its thermal stability is increased significantly by 23 degrees C. hER alpha is a nuclear receptor (NR) family member. In NRs, protein function is allosterically regulated by its interplay with small molecule effectors and the interaction with coregulatory proteins. The in-depth characterization of ERPRS* shows that these cooperative effects are fully preserved despite that 10% of all residues were substituted(.) Crystal structures reveal several salient features, i.e. the introduction of a tyrosine corner in a helix-loop-helix segment and the formation of a novel surface salt bridge network possibly explaining the enhanced thermal stability. ERPRS* shows that prior successes in computational approaches for stabilizing proteins can be extended to proteins with complex allosteric regulatory behaviors as present in NRs. Since NRs including hER alpha are implicated in multiple diseases, our ERPRS* variant shows significant promise for facilitating the development of novel hER alpha modulators.

Automated summary

The application of PROSS server algorithms enabled the development of variant ERPRS* with 24 amino acid substitutions, leading to improved production rates, crystallization, and thermal stability. Despite 10% amino acid substitutions, ERPRS* preserved the allosteric regulatory behaviors of nuclear receptors, suggesting promise for developing novel modulators targeting hER alpha.