Descriptions of materials, metrological methods, computational methods, and supplementary results. Figures of HDX-MS publications and citations versus publication year, histogram of peptide sequence lengths, sequence coverage maps, performance of instrumentsoftware configurations, repeatability plots, %E corrected peptide t HDX versus log 10 (t HDX ) for eight peptides. Tables of instrumentation,software, peptide search methodology, and operating conditions of proteolytic, chromatographic components, and effects of peptide charge on deuterium uptake (PDF)

Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site. Cytochrome P450 46A1 (CYP46A1)2 is a heme-containing, microsomal monooxygenase that catalyzes regio-and stereoselective hydroxylation of cholesterol to 24(S)-hydroxycholesterol (24HC) (1, 2). Cholesterol 24-hydroxylation represents the major mechanism for cholesterol elimination from the mammalian brain and accounts for ϳ75-85% of cerebral cholesterol removal in humans and ϳ40 -50% of cholesterol disposal in mice (3-5). Studies in vitro and in vivo revealed that CYP46A1 can bind compounds other than cholesterol, including sterols and some marketed drugs (6 -13), with compound binding leading to enzyme inhibition (8, 10). Unexpectedly, several inhibitory pharmaceuticals were also found to act, at specific concentrations, as enzyme activators enhancing CYP46A1-mediated 24HC production (7, 13). Both increase and decrease of CYP46A1 activity have therapeutic potential (14); therefore, we began to ascertain the molecular basis of CYP46A1-drug interactions. Spectroscopic and crystallographic characterizations of substrate-free, substrate-bound, and seven CYP46A1-drug complexes established that inhibitory drugs bind to the enzyme active site because it is plastic and can accommodate compounds of different sizes and shapes because of a ligand-induced conformational fit (7, 9 -12). In contrast, binding of the activating drugs is not yet fully understood but began to be elucidated for efavirenz (EFV), a Food and Drug Administration-approved anti-HIV medication. We established that, both in vitro and in vivo (in mice), the dependence of CYP46A1 activity on EFV concentration represents a bell-shaped curve with CYP46A1 activation at low EFV concentrations and inhibition at higher drug concentrations (13). We also generated a model of CYP46A1 activation by EFV (13) according to which CYP46A1 has two separate sites: the allosteric site located on the cytosolic surface of the molecule and the active site embedded in the lipid bilayer. At low concentr...