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

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.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

“…9 C ). However, this salt bridge is neither conserved across herpesviruses nor appears to be subfamily-specific although surface-located salt bridges have been shown to increase the structural stability of proteins ( 36 , 37 ).…”

pH and the Breast Cancer Recurrent Mutation D538G Affect the Process of Activation of Estrogen Receptor α

“…Previous studies also showed similar improvements in thermostabilization of the activated ER. 50,51 Although the holo ER wt has similar T m values at both pHs, apo ER wt presents different values at pH 6.0 (T m = 43) and pH 8.0 (T m = 48). Such a result may be attributed to the stronger tendency of H12 to reach its closed state at the higher pH, even in the absence of E2.…”

Efficient Exploration of Sequence Space by Sequence-Guided Protein Engineering and Design

“…This combination of sequence-based and structure-based design has been successfully implemented in two computational tools, PROSS and FireProt, that aim to redesign proteins for increased thermostability and expression. ,,,− PROSS uses the observed amino acid frequencies at each position in a multiple-sequence alignment to define a set of “allowed” substitutions in the target protein on the basis of their occurrence in homologous proteins (Figure ). Allowed substitutions that have a stabilizing effect on protein structure are predicted using structure-based energy calculations, and mutually compatible combinations of these substitutions are then predicted by combinatorial protein design in Rosetta, yielding a small number of designed sequences for experimental testing with substitutions at ≲10% of positions.…”