Deciphering allosteric coupling between substrate binding and redox partner recognition in rat CYP24A1, a Vitamin‐D C‐24 hydroxylase

Abstract

The mitochondrial cytochrome P450 24A1 (CYP24A1) is necessary for the deactivation of bioactive vitamin-D (1,25(OH)2D3) by side-chain hydroxylation. The enzyme requires molecular recognition by its cognate redox partner adrenodoxin (Adx), which houses an iron-sulfur cluster and provides two electrons for the hydroxylation step to achieve the reduction of heme iron in CYP24A1. Dysregulation of CYP24A1 may lead to idiopathic infantile hypercalcemia, characterized by elevated calcium, failure to thrive, vomiting, dehydration, and nephrocalcinosis. We have previously used solution nuclear magnetic resonance (NMR) to demonstrate the long-range allostery of CYP24A1 in response to redox partner recognition. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1–Adx complex. We report that differential chemical cross-linking internal to CYP24A1 points toward an Adx-induced conformational change that perturbs the F and G α-helices, which are required for substrate binding. The combined presence of Adx with 1,25(OH)2D3 induces a change in this cross-linking pattern, consistent with an open-to-closed change in the enzyme. Both outcomes suggest conformational coupling in the CYP24A1 structure in response to Adx and 1,25(OH)2D3. Moreover, the modeled complex suggests that a semi-conserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. This insight leads us to a revised hypothesis in which conformational coupling is also important in determining site-specific hydroxylation of the CYP24A1 substrate. Taken together, these findings contribute toward our understanding of Adx recognition in a critical vitamin-D inactivating enzyme and provide broader insight regarding the variability inherent in CYP–Adx interactions.