The N-Terminus of Human Sulfotransferase 2B1b─a Sterol-Sensing Allosteric Site.

ABSTRACT

Among human cytosolic sulfotransferases, SULT2B1b is highly specific for oxysterols─oxidized cholesterol derivatives, including nuclear-receptor ligands causally linked to skin and neurodegerative diseases, cancer and atherosclerosis. Sulfonation of signaling oxysterols redirects their receptor-binding functions, and controlling these functions is expected to prove valuable in disease prevention and treatment. SULT2B1b is distinct among the human SULT2 isoforms by virtue of its atypically long N-terminus, which extends 15 residues beyond the next longest N-terminus in the family. Here, in silico studies are used to predict that the N-terminal extension forms an allosteric pocket and to identify potential allosteres. One such allostere, quercetin, is used to confirm the existence of the pocket and to demonstrate that allostere binding inhibits turnover. The structure of the pocket is obtained by positioning quercetin on the enzyme, using spin-label-triangulation NMR, followed by NMR distance-constrained molecular dynamics docking. The model is confirmed using a combination of site-directed mutagenesis and initial-rate studies. Stopped-flow ligand-binding studies demonstrate that inhibition is achieved by stabilizing the closed form of the enzyme active-site cap, which encapsulates the nucleotide, slowing its release. Finally, endogenous oxysterols are shown to bind to the site in a highly selective fashion─one of the two immediate biosynthetic precursors of cholesterol (7-dehydrocholesterol) is an inhibitor, while the other (24-dehydrocholesterol) is not. These findings provide insights into the allosteric dialogue in which SULT2B1b participates in in vivo and establishes a template against which to develop isoform-specific inhibitors to control SULT2B1b biology.