Structural Biology of Urothelial Membranes

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The apical surface of bladder epithelium is covered by rigid-looking plaques consisting of hexagonally packed crystalline arrays of 16 nm protein particles made up of four major uroplakins (UPs). These urothelial plaques serve as an effective permeability barrier, and may play a role in the reversible adjustment of the urothelial apical surface area during different phases of the micturition cycle, In addition, the attachment of uropathogenic type 1-piliated E. coli to their uroplakin Ia receptor can cause urothelial cytoskeletal rearrangement, apoptosis and bacterial invasion. The goal of this project is to understand the structural basis of urothelial plaque functions. Based on our recently obtained 10 Angstrom resolution cryo-EM structure of the 16 nm mouse urothelial particles, we hypothesize that (i) the two uroplakin pairs, i.e., UPIa/II and UPIb/III, occupy the inner and outer six subdomains of the 16 nm particle, respectively, and that (ii) the relatively flexible structure of the 16 nm uroplakin particle can mediate transmembrane signal transduction through conformational changes. To test these hypotheses and to further understand the structure-function relationship of the urothelial plaques, we will perform three series of studies that will: (1) visualize the individual transmembrane helices of the uroplakins by obtaining a cryo-EM structure of the 16nm particle at the resolution range of 7 Angstroms dock the atomic models of UPIa and Ib into the cryo-EM density maps thus improving the 3D modeling, and prepare 3D crystals of uroplakins aiming at solving uroplakin strucure to atomic resolution; (2) localize in the 3D architecture of the 16 nm particle several uroplakin moieties using Fab fragments and specific lectins as the probes; and (3) study the possible bacterial binding-induced conformational changes of the 16 nm uroplakin particle by comparing the 3D cryo-EM structures of the particle in the presence and absence of saturating amounts of the bacterial adhesin FimH. 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.