Projection Structure by Single-Particle Electron Microscopy of Secondary Transport Proteins GltT, CitS, and GltS

Abstract

The structure of three secondary transporter proteins, GltT of Bacillus stearothermophilus, CitS of Klebsiella pneumoniae, and GltS of Escherichia coli, was studied. The proteins were purified to homogeneity in detergent solution by Ni 2þ -NTA affinity chromatography, and the complexes were determined by BN-PAGE to be trimeric, dimeric, and dimeric for GltT, CitS, and GltS, respectively. The subunit stoichiometry correlated with the binding affinity of the Ni 2þ -NTA resin for the protein complexes. Projection maps of negatively stained transporter particles were obtained by single-particle electron microscopy. Processing of the GltT particles revealed a projection map possessing 3-fold rotational symmetry, in good agreement with the trimer observed in the crystal structure of a homologous protein, Glt Ph of Pyrococcus horikoshii. The CitS protein showed up in two main views: as a kidney-shaped particle and a biscuit-shaped particle, both with a long axis of 160 A ˚. The latter has a width of 84 A ˚, the former of 92 A ˚. Symmetry considerations identify the biscuit shape as a top view and the kidney shape as a side view from within the membrane. Combining the two images shows that the CitS dimer is a protein with a strong curvature at one side of the membrane and, at the opposite side, an indentation in the middle at the subunit interface. The GltS protein was shaped like CitS with dimensions of 145 A ˚Â 84 A ˚. The shapes and dimensions of the CitS and GltS particles are consistent with a similar structure of these two unrelated proteins.

Projection Structure of the Secondary Citrate/Sodium Symporter CitS at 6 Å Resolution by Electron Crystallography

“…The observed elliptical shape of the asymmetric unit is consistent with the low-resolution projection structure derived from single-particle analysis of detergent-solubilized dimeric CitS. 38 As expected, the crystal projection map shows a remarkably smaller outer dimension of the molecule (9.6 nm × 5.2 nm) than seen for the detergent-surrounded particles previously observed (16 nm × 8.4 nm). 38 The obtained dimensions of the dimeric CitS symporter are similar to those of other secondary transporters such as the bacterial chloride channel ClC 51 or the Na + /H + exchangers NhaA 52 and NhAP1.…”

“…38 As expected, the crystal projection map shows a remarkably smaller outer dimension of the molecule (9.6 nm × 5.2 nm) than seen for the detergent-surrounded particles previously observed (16 nm × 8.4 nm). 38 The obtained dimensions of the dimeric CitS symporter are similar to those of other secondary transporters such as the bacterial chloride channel ClC 51 or the Na + /H + exchangers NhaA 52 and NhAP1. 50 The 2D crystal arrangement shows CitS in a dimeric form, which corroborates the dimerization findings from Blue Native PAGE, single-particle electron microscopy (EM), and fluorescence spectroscopy.…”

“…Additionally, we found the wider planar tubes to be much better ordered than crystals with smaller diameters. Hence, CitS may have the intrinsic tendency to form tubes within a small size range, which might be due to protrusions in the molecule, as found by Moscicka et al 38 Interestingly, crystals grown in acetate buffer were consistently better ordered compared to crystals in citrate buffer with otherwise identical conditions. This might be related to a higher conformational flexibility of CitS in the presence of its substrate citrate as predicted from the model of "alternating access".…”

“…50 The 2D crystal arrangement shows CitS in a dimeric form, which corroborates the dimerization findings from Blue Native PAGE, single-particle electron microscopy (EM), and fluorescence spectroscopy. 38,39 However, it is still unclear whether the monomeric or the dimeric CitS forms the functional unit. In comparison, most available structures from secondary transporters so far exhibit higher oligomeric states such as dimers (e.g., NhaP1) and trimers (e.g., the H + /galactose symporter GalP).…”

“…37 Structural and biophysical studies suggested detergent-solubilized and purified CitS to exist as an elliptical shaped homodimer. 38,39 Krupnik et al assigned the interface between the two monomers to the short axis of the elongated particle, leaving the long axis for the interface between the N-and Cterminal domains within one monomer (Fig. 1b).…”