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.
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.
After extracting a single asymmetric unit from the map, the X-ray model published by Lu et al. (2009, pdb 3H90) was docked to the map. This required significant rearrangements to the transmembrane domain, probably reflecting a conformational changes related to the absence on Zn2+ in our new structure.
X-ray structure placed in EM map does not fit very well
X-ray structure refitted to EM map
Indeed, surface rendering of the transmembrane domain reveals that there is access to the transport sites from the cytoplasmic side of the membrane in the EM structure. This contrasts with the X-ray structure, which shows access from the extracellular side of the membrane. These observations suggest that YiiP adopts two different conformations in the alternating access model for transport.