Zn2+ transporter YiiP
| Zinc is essential as a cofactor for a variety of enzymes and transcription factors and plays a role in various biological processes ranging from gene expression to the immune response. Abnormal levels of Zn2+ plays a role in many diseases, including Alzheimer’s and type-2 diabetes. YiiP is a member of the Cation-Diffusion Facilitator (CDF) family and it catalyzes the exchange of Zn2+ and H+ across the membrane. Previous X-ray crystallographic studies (Lu et al. 2007, 2009) produced atomic structures for Yiip with Zn2+ bound at 4 different sites. This structure reveals a membrane-bound domain with six transmembrane helices and a cytoplasmic domain with a fold resembling the metallochaperones used to shuttle transition ions like Cu+ through the cytoplasm. The crystal structure also shows YiiP forming a homo-dimer with Zn2+ mediating interactions between the cytoplasmic domains, but a large distance between the transmembrane domains. |
X-ray structure of YiiP (Lu & Fu, 2007,2009) |
Tubular crystals of the Q8E919 protein, We have expressed a Yiip homolog (Q8E919) from Shewanella oneidensis and purified it with DM. Crystals were found after extensive screening with a home-made 96-well dialysis chamber. For crystallization, different lipid compositions, the pH's,lipid-to-protein ratios and salt compositions were tested. The best crystals were found at pH=7 with DOPG lipids solubilized in DDM at room temperature. Tubular crystals grow over a large range of lipid-to-protein ratios (0.25 to 1.5) and all attempts to produce planar crystals were unsuccessful, supporting the idea that protein-protein crystal contacts dictate the final morphology of the crystals. Nevertheless, to crystal forms have been observed with different diameters, as seen in the image to the right. |
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For 3D reconstruction, both Fourier-Bessel and the Iterative Helical Real-Space Reconstruction (IHRSR) methods have been used to obtain the 3D map of the structure. Fourier-Bessel reconstruction has relied on EMIP, a graphical user interface developed in our lab and interfacing with well-known algorithms developed by Beroukhim & Unwin (1997). The IHRSR reconstruction was done with SPARX in collaboration with Juoehi & Penczek. Indexing of helical symmetry shows that tubes have four-fold symmetry with helical parameters of dφ = -42.3 ° and dz = 17Å. The ~13Å structure a narrow cylinder (inner radius <50 Å) with tight packing of the proteins in the lipid bilayer. The C-terminal domain of the proteins protrudes from the outer surface. Docking of the X-ray model published by Lu et al. (2009, pdb 3H90) is currently underway; however, it appears that the model must undergo significant rearrangements to match the obtained EM map, probably reflecting conformational changes associated with the absence on Zn2+ in our new structure.
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