Cryo-EM Structure and Function of the Human BEST1 Channel Complexed with a Metabolic Enzyme

초록

The Bestrophin (BEST) channels belong to a class of calcium-activated chloride (Cl-) channels that are activated by an increase in cytoplasmic [Ca2+]. In the human genome, four BEST channel paralogs (BEST1 to BEST4) have been identified. These human BEST channel paralogs (hBEST1 to hBEST4) share approximately 60% sequence identity in the N-terminal region of around 370 amino acids, but only retain 6% to 19% sequence identity in the remaining C-terminal region, which is 150-200 amino acids long. Similarly, there are distinct sequence differences between the human and mouse BEST1 orthologs. Our recent research has demonstrated that the marginally conserved C-terminal region plays critical roles in regulating calcium sensitivity, cellular expression, and plasma membrane targeting of both human and mouse BEST1 channels. To gain a deeper understanding of the molecular mechanism underlying the functional differences between the human and mouse BEST1 channels, we conducted immunoprecipitation-mass spectrometry (IP-mass) analysis to identify proteins that specifically interact with the hBEST1 C-terminal region. Interestingly, we discovered that an enzyme called PYCR3, which is involved in proline metabolism in the cytosol, serves as a binding partner for hBEST1. This interaction was further confirmed through co- immunoprecipitation and size-exclusion chromatography experiments. Moreover, we successfully determined the structure of the hBEST1-PYCR3 complex at a resolution of ~3 Å. In the complex structure, PYCR3 forms a decameric assembly and attaches to the cytosolic face of the hBEST1 pentamer. Notably, compared to the closed channel structure, the transmembrane domain of hBEST1 undergoes rotation in the complex structure, suggesting that the interacting enzyme may facilitate the opening of the hBEST1 channel. Indeed, the co-expression of PYCR3 led to an increase in the current density of hBEST1 channels, while the knockdown of endogenous PYCR3 reduced BEST1 channel activity. We are currently investigating whether the enzymatic activity can regulate the electrophysiological characteristics of hBEST1 channels, or vice versa.