Zinc transporters play a central role in regulating zinc homeostasis in cells. Zinc efflux transporters can discriminate Zn2+ from similar cytosolic cations and respond to its fluctuations around a homeostatic set point by pumping excess amounts out of the cytoplasm against steep transmembrane Zn2+ concentration gradients. At present, we have no knowledge of the structure of any zinc transporter. Our long-term goal is to understand the structural basis for selective binding and energized movement of zinc ions in mammalian zinc transporters. This proposed study will focus on cation diffusion facilitators (CDFs) that represent a major family of zinc efflux pumps in diverse organisms from bacteria to mammals. Specifically, we will use x-ray crystallographic- and biochemical-approaches to explore how the zinc affinity, selectivity and mobility are built into the structure of YiiP, a prototypic CDF protein from Escherichia coli.
Two specific aims are proposed; (1) To determine the crystal structure of YiiP to reveal a molecular architecture for a model CDF, and, (2) To elucidate the structural basis for binding and transport of Zn2+. This will involve (a) interpreting the crystal structure based on existing biochemical data, (b) determining the functional roles of the observed metal-binding sites in the crystal structure, (c) analyzing the coupling between metal binding and YiiP deprotonation, and, (d) characterizing the kinetics of YiiP motions in response to metal binding. The proposed structural and biochemical analyses will lead to the first x-ray crystallographic solution of a zinc transporter structure and afford a detailed kinetic model for the conformational changes in YiiP that drive the uphill pumping of Zn2+ against an opposing downhill flow of H+. These results will open the door to homology modeling of human CDF structures, thereby setting the stage for structure-based drug design targeting ZnT- 3, a homologous human CDF that is implicated in the pathogenesis of Alzheimer's disease. ? ? ?
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