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.

YiiP X-ray structure

X-ray structure of YiiP (Lu & Fu, 2007,2009)
Black lines indicate the membrane surfaces.

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.

YiiP tubes

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.

FFT of YiiP tubular crystals
YiiP Helical Reconstruction

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.

YiiP Xray fit to EM map
X-ray structure placed in EM map does not fit very well
YiiP refitted to EM map
X-ray structure refitted to EM map
YiiP Xray fit to EM map
Use of the Titan Krios Microsope and LEGINON image processing led to a much higher resolution structure: 4.1 A overall with the membrane domain at 3.5 A or higher. This allowed us to build an atomic model.
YiiP refitted to EM map
One surprise is that strong density was present at all the Zn sites even though Zn was absent during crystallization.


We made cysteine substitutions to four residues at the dimer interface within the membrane (shown in yellow above) and tested the ability of these mutants to transport Zn, after formation of di-sulfide bonds to prevent the scissoring motion that is suggested by comparisons with the X-ray structure.

Alteranating Access model for transport

Based on this result, we produced a hybrid model for the outward-facing conformation which maintains the interdimer interface, but allows movements of the helical bundles that compose each monomer