Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ~3.0Å structure contains 14 transmembrane helices in an inward facing conformation with a core structure of inverted repeats of 5 TM helices (TM2-TM6 and TM7-TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

The Na ؉ -Ca 2؉ exchanger plays a central role in cardiac contractility by maintaining Ca 2؉ homeostasis. Two Ca 2؉ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca 2؉ binding to these regulatory domains activates the transport of Ca 2؉ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca 2؉ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca 2؉ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca 2؉ ions in primary and secondary Ca 2؉ sites. In the absence of Ca 2؉ , Lys 585 stabilizes the structure by coordinating two acidic residues (Asp 552 and Glu 648 ), one from each of the Ca 2؉ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca 2؉ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca 2؉ site completely remove Ca 2؉ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na ؉ -Ca 2؉ exchanger.calcium binding ͉ calcium regulation

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C in ) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C out ) to C in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescencedetection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C out topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.