Structural Biology of Urothelial Membranes

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The apical surface of mammalian urothelium is covered by rigid plaques (also known as asymmetric unit membrane or AUM) that are hexagonally packed crystalline arrays of i6-nm particles consisting of four uroplakins (UPs). These uroplakin plaques contribute to the urothelial permeability barrier function, and one of the uroplakins, UPIa, can serve as the receptor for the type 1-fimbriated uropathogenic E. coli (UPEC). UPEC gains a foothold in the urinary tract by binding, via its adhesin FimH, to the urothelial receptor. During the last grant period, we obtained a 6A resolution cryo-EM structure of the 16-nm uroplakin particle and showed that FimH binding can induce large conformational changes of the extracellular domains as well as the transmembrane helices of the uroplakins. These results suggest that bacterial attachment can induce a novel mechanism of transmembrane signal transduction leading to bacterial invasion and establishment of urinary tract infection (UTI), one of the most common infectious diseases. In the next grant period, we will continue our efforts in studying the structure-function relationship of the 16-nm particle and the mechanism of bacterial binding-induced signal transduction. In Aim one, we will establish an atomic model of the i6-nm particle by performing an EM density-aided model building with the help of cryo-EM of improved resolution and domain structures of uroplakins that we can dock into the electron densities. We will obtain full-length recombinant uroplakin molecules for 3D crystallization, aiming at atomic resolution structures of the uroplakins. In Aim two, we will carry out an electron tomographic 3D visualization of the AUM-cytoskeleton connections in normal, in mechanically stretched, and in infected bladder. We will also investigate the identities of the proteins that form the bridge interconnecting the AUM and the cytoskeleton. Our results should lead to a better understanding of the structural bases of urothelial plaque function, and of the possible roles of urothelial plaques in urinary tract infection.