Structure function studies of human estrone sulfatase as a target for hormone-dependent breast cancer therapeutics

Estrone Sulfatase (ES) is a membrane bound enzyme of the endoplasmic reticulum (ER) that catalyzes the release of unconjugated estrone (E1) from the body pool of estrone sulfate. E1 is subsequently reduced to 17β-estradiol (E2) by type 1 17β-Hydroxysteriod Dehydrogenase. Nearly 50% of all breast cancer cases are hormone dependent. High levels of E2 have been linked to growth and proliferation of breast tumors.; The full-length, fully active ES (562 amino acids) was purified to homogeneity from the microsomal fraction of human placenta and stabilized in suitable detergent conditions. Diffraction quality crystals were obtained with methyl-2,4-pentanediol as the precipitant, in β-D-octylglucopyranoside detergent. X-ray diffraction data from ES crystals frozen in liquid nitrogen were collected to 2.6Å resolution at the Cornell High Energy Synchrotron Source. The structure was solved by the Single Isomorphous Replacement Anomalous Scattering (SIRAS) method with the aid of a mercury derivative, followed by solvent flattening. The resulting high quality electron density map enabled tracing of nearly 80% of the molecule. A molecular replacement solution using the structure of soluble Aryl Sulfatase A as the search model, greatly aided tracing of poorly defined regions. The atomic model was refined using the CNS software package.; The structure of ES is characterized by two highly hydrophobic anti-parallel alpha helices, each approximately 40Å long, clearly visible in the SIRAS map. Presumably, these helices traverse the ER membrane, thereby anchoring the enzyme to the membrane surface. Thus, the otherwise spherical molecule with an average diameter of about 55Å has an overall appearance of a “mushroom”. The active site resembles those of other sulfatases, and is located in an inner cavity. The entrance to the active site rests at or near the membrane surface, in close proximity to the confluence of the transmembrane helices. This architecture suggests an active participation of the ER membrane in steroidal substrate capture and/or product release.; The ES structure paves the way towards further investigation into rational design of specific inhibitors that could be used as drugs for prevention and treatment of hormone dependent breast cancer.