A PROSS-designed extensively mutated estrogen receptor \u03b1 variant displays enhanced thermal stability while retaining native allosteric regulation and structure

Mark Kriegel,Sewar Alkhashrom,Jutta Eichler,Hanna J Wiederanders,Yves A Muller

doi:10.1038/s41598-021-89785-1

Mark Kriegel, Sewar Alkhashrom + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-89785-1

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Journal: Scientific Reports	Publication Date: May 18, 2021
Citations: 15	License type: open-access

Affiliation: University of Erlangen-Nuremberg

Abstract

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 (hERα) 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 °C. hERα 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 hERα are implicated in multiple diseases, our ERPRS* variant shows significant promise for facilitating the development of novel hERα modulators.

Highlights

Protein stability limitations often hamper the exploration of proteins as drug targets
While agonist binding promotes the interaction of the ligand-binding domain (LBD) with coregulatory proteins, such as for example the interaction of hERα with the steroid receptor coactivator-2 (SRC-2) protein, binding of antagonists leads to a rearrangement of so-called helix 12 (H12), and this rearrangement precludes any further interaction with coregulators (Supplementary Fig. S1)[2,3,4]
We show that E RPRS* yields higher production rates in E. coli and displays a significant increase in thermal stability of ~ 23 °C

Summary

Introduction

Protein stability limitations often hamper the exploration of proteins as drug targets. Small molecule effectors acting as either agonists or antagonists bind to an identical pocket in the LBD of NRs. While agonist binding promotes the interaction of the LBD with coregulatory proteins, such as for example the interaction of hERα with the steroid receptor coactivator-2 (SRC-2) protein, binding of antagonists leads to a rearrangement of so-called helix 12 (H12), and this rearrangement precludes any further interaction with coregulators (Supplementary Fig. S1)[2,3,4]. While agonist binding promotes the interaction of the LBD with coregulatory proteins, such as for example the interaction of hERα with the steroid receptor coactivator-2 (SRC-2) protein, binding of antagonists leads to a rearrangement of so-called helix 12 (H12), and this rearrangement precludes any further interaction with coregulators (Supplementary Fig. S1)[2,3,4] These structural rearrangements have been shown in detail for hERα but details may differ in other human nuclear r eceptors[5]. This procedure allows for substitutions to be included in the final design that are predicted to be neutral or singly negative according to the Rosetta calculations and are favored by p hylogeny[17,18]

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