Structure of the Borate Transporter Bor1p by cryo-EM

Boron is an essential micronutrient for plants and animals. Transport of boron regulates uptake and protects against high levels of boron in plants and yeast. The yeast boron transporter Bor1p belongs to the SLC4 Anion Exchanger family of transporters that also includes the human chloride/bicarbonate exchanger. We have overexpressed and purified the Bor1p orthologue from Saccharomyces mikatae using Saccharomyces cerevisiae as a host. Helical crystals were produced by reconstituting the transporter together with cardiolipin and electron micrographs of frozen-hydrated tubes were recorded with a field emission gun at 200 kV using a direct electron detector. Crystals adopted two different helical symmetries from which two independent reconstructions were produced from 75 particles at a resolution of ∼7 A using a Fourier-Bessel approach. In order to interpret the dimeric densities revealed by the reconstructions, we built a homology model for Bor1p based on the crystal structure of the uracil transporter (UraA), which is the closest relative to Anion Exchangers within the APC superfamily of transporters. Initial homology models were produced from 10 alternative alignments of these distantly related proteins. The alignments were refined by mapping sequence conservation onto these 3D structures. The best model was then fitted to the cryo-EM map by adjusting the position of individual helices and the resulting structure was equilibrated in a lipid bilayer for 200 ns using molecular dynamics. The resulting structure suggests conformational changes relative to UraA in which helices at the dimer interface are tilted relative to the transporter domain, thus providing access to the substrate binding site from the extracellular side of the membrane. Comparison of our Bor1p structure with that from UraA are likely to reflect the structural changes that accompany the alternating access mechanism employed by this family of transporters.