Structural Basis for Mechanism of Secondary Transporters

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DESCRIPTION (provided by applicant): At least 20 percent of the genomes sequenced thus far encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and signal transduction. Many are important with regard to human disease (eg. depression, diabetes, drug resistance), and many drugs are targeted to membrane transport proteins (eg. fluoxetine and omeprazole). However, the number of crystal structures of membrane proteins, particularly ion-coupled transporters, is very limited. Recently, an inward-facing conformer of the Escherichia coil lactose permease (LacY) was solved at about 3.5 A in collaboration with So Iwata's group at Imperial College London. This intensively studied member of the Major Facilitator Superfamily (MFS), which contains over 1000 members, is composed of two symmetrical 6-helix bundles with a large internal cavity containing bound sugar, which is open to the cytoplasm only. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism is proposed in which the binding site is alternatively accessible to either side of the membrane. The specific aims proposed here are to test this hypothesis by constructing and obtaining a crystal structure(s) in the outward-facing conformation(s). Since other members of the MFS exhibit a similar overall structure, as indicated by modeling studies, we also propose to determine structures for certain LacY homologues. Insights obtained from the current structure, as well as a variety of biochemical and biophysical techniques, will be used to rationally design and characterize mutants in the outward-facing conformation(s) prior to crystallization trials and structure determination. We will also obtain higher resolution of the current structure in the near future, and we expect the other structures proposed to be obtained within the time period planned. X-ray structures of these transport proteins in multiple conformations will have important impact on biology and medicine.