Abstract
Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.
Highlights
Oxidative hydroxylation of the steroid hormone estrogen, which results in the formation of catechol estrogens (CEs1; Fig. 1), is the first step in estrogen catabolism
The conversion of ATP to cAMP in mammals is mediated by members of the class III adenylyl cyclase family (AC; E.C. 4.6.1.1), which in humans comprises nine trans-membrane AC enzymes and one soluble AC [8, 9]. tmACs are regulated by heterotrimeric G proteins in response to the stimulation of G protein-coupled receptors and play a key role in the cellular response to extracellular signals [10]. sAC, in contrast, is insensitive to G proteins and the additional tmAC regulator calmodulin as well as to the non-physiological diterpenic activator forskolin [11, 12]
The adenylyl cyclase activity assays showed that CE inhibited the generation of cAMP by these membrane preparations in a concentrationdependent manner (Fig. 2C and Supplemental Fig. 1, available in the on-line version of this article)
Summary
Oxidative hydroxylation of the steroid hormone estrogen, which results in the formation of catechol estrogens (CEs1; Fig. 1), is the first step in estrogen catabolism. CEs elicit some physiological responses via the classical estrogen receptor, other biological CE effects are not associated with activation of this receptor [5, 6]. In the latter regard, it has long been known that CEs exert effects in the cAMP signaling system, as shown by their inhibition of cAMP accumulation in the hypothalamus [7]. SAC, in contrast, is insensitive to G proteins and the additional tmAC regulator calmodulin as well as to the non-physiological diterpenic activator forskolin [11, 12]. TmACs and sAC regulate a diverse set of essential biological processes such as differentiation and gene transcription
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