Zinc transporters play a central role in metal metabolism and homeostasis in all living organisms. Our long- term goal is to understand the structure and mechanism of zinc transporters and their functional regulation by metal ions, lipids, and drugs. This is the second competitive renewal of a continuing research project focusing on zinc-efflux transporters from the cation diffusion facilitator (CDF) family. In the current budget period, we solved the crystal structure of YiiP, a prototypic CDF ortholog from E. coli. The crystal structure reveals a Y- shaped architecture that is uniquely oriented in the membrane with an active-site situated near the protein-lipid interface. Despite our existing crystallographic snapshots, a structure-based transport model still is lacking, and the relevance of the YiiP crystal structure to its native structure in the membrane is uncertain. Further, YiiP is a two-modular membrane transporter with a cytoplasmic domain for zinc sensing, and a transmembrane domain that transports zinc ions in response to fluctuation of cytoplasmic zinc concentrations. The conformational changes underlying the allosteric regulation of zinc transport are yet to be explored. In the next budget period, we will use a combination of low-resolution biochemical techniques and high-resolution crystallographic analysis to address three questions: (i) how does YiiP move a cytoplasmic zinc ion across the membrane barrier, (ii) how is the active-site reshaped by surrounding lipids, and, (iii) how do conformational changes in YiiP enable a regulated zinc-transport activity tunable to the cytoplasmic zinc concentration? Accordingly, we propose three specific aims: (i) developing a mechanistic model for zinc transport, (ii) evaluating YiiP conformations in the membrane, and, (iii) defining the YiiP structure in action. During the past two budget cycles, we have developed functional assays for zinc transport with millisecond time-resolution, and obtained structural information on a CDF ortholog at atomic resolution. In the next budget period, we will explore the inner workings of CDFs in atomic detail (aim-1), in native membrane environments (aim-2), and in action (aim-3). PUBLIC HEALTH RELEVANCE: Zinc efflux transporters in the cation diffusion facilitator (CDF) family are important molecular markers and active drug targets for diabetes and Alzheimer's disease. The fundamental knowledge gained through the proposed structure-function study will illuminate the functional consequences of human CDF polymorphisms, thereby providing the basis for disease-risk assessment by genetic screening. Further, homology modeling of human CDF orthologs will be instrumental to structure-guided drug discovery efforts.