Structural Basis of SERCA Inhibition by Derivatives of di-tert-butylhydroquinone Revealed by X-ray Crystallography

The small molecule di-tert-butylhydroquinone (BHQ) is a potent inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). It inhibits the enzyme by stabilizing it in its E2 conformation and thereby preventing the transition to the E1 state. To define the structural determinants underlying BHQ inhibition, we examined ten BHQ derivatives with systematic modifications of polar and hydrophobic substituents of different shapes and sizes. Inhibitory potencies were determined in ATPase activity assays, and crystal structures of SERCA1a in complex with eight of the ten derivatives were obtained at resolutions of 2.6 Å or better, aided by co-crystallization of thapsigargin to stabilize the E2 state. The structures revealed that the compounds bind in a pocket formed by transmembrane helices M1-M4, in which the ligand is stabilized by CH-π interactions between the central benzene ring and Leu61 and Pro312, by hydrogen bonds - preferentially involving Asp59 - and by extensive van der Waals contacts mediated by two butyl substituents. Analysis of the SERCA/inhibitor poses showed that while at least one hydrogen bond is required for binding, inhibitory potency correlates strongly with the number and quality of van der Waals contacts mediated by appropriately positioned alkyl groups. Compounds lacking alkyl substituents or containing oversized or mispositioned groups displayed markedly reduced potencies. Although no derivative exceeded BHQ in inhibitory activity, structural analysis suggests that repositioning hydroxy groups on the benzene ring or modifying alkyl substituents may further optimize binding affinity and thus inhibitory potency. Together, these results define the key molecular features of SERCA inhibition by BHQ analogs and suggest strategies for further optimization of this inhibitor class.