Conformational heterogeneity of the aspartate transporter GltPh

Abstract

Glt Ph is a Pyrococcus horikoshii homotrimeric Na + -coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the conformational changes underlying transport by Glt Ph using EPR spectroscopy. The trimerization domains form a rigid scaffold, whereas the transporting domains sample multiple conformations, consistent with large-scale movements during the transport cycle. Binding of substrates changed the occupancies of the different conformational states, but the domains remained heterogeneous. The membrane environment favored conformations different from those observed in detergent micelles, but the transporting domain remained structurally heterogeneous in both environments. We conclude that the transporting domains sample multiple conformational states with substantial occupancy regardless of the presence of substrate and coupling ions, consistent with equilibrium constants close to unity between the observed transporter conformations. npg © 2013 Nature America, Inc. All rights reserved.nature structural & molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 2 1 1 a r t i c l e s of 160 K to qualitatively extract information on interspin distances <1.8 nm (ref. 14). We analyzed Glt Ph both solubilized in detergent micelles and reconstituted in proteoliposomes and compared the results from each experiment type. The trimerization domain is a stable scaffoldIntroduction of a spin label at a single position in Glt Ph allows for the measurement of the distance between the protomers of the homotrimeric protein. We constructed two trimerization-domain mutants by replacing Thr166 (located in the cytoplasmic end of transmembrane helix 4) and Val176 (located in the cytoplasmic end of helix 5) with an MTSL-modified cysteine (R1). Simulations on the available crystal structures indicated that these spin labels are >1.8 nm apart and therefore suitable for DEER measurements 15 . We did not observe spectral broadening in the CW EPR measurements, which further supports the notion that the spin labels were >1.8 nm apart (Supplementary Fig. 1). We recorded spectra of the apoprotein and of the protein in the presence of saturating concentrations of Na + alone or Na + and aspartate. In agreement with previous biochemical and structural data 12,13 , the trimerization domain did not show any large-scale conformational changes upon addition of coupling ions or substrate (Fig. 2). We observed two sharp peaks (centered around 2.8 nm and 3.8 nm for T166R1 and 2.6 nm and 3.4 nm for V176R1) in the interspin distance distributions for proteins in the presence and absence of substrates. The measured distances were in agreement with the interprotomer distances calculated on the basis of the crystal structures (Table 1 and Fig. 2).Because Glt Ph is a trimer, each mutation resulted in the presence of three cysteines per protein complex, which could result in a cons...

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

“…1D, Upper), consistent with crystal structures of extreme states ( Fig. S3A) (6,7), sm-FRET (8,16,17), and electron paramagnetic resonance (EPR) (19,20) studies. Kymographs converted to idealized traces of transitions between outward-and inward-facing conformations (Fig.…”

Crystal structure of a substrate-free aspartate transporter

“…3). This hypothesis is supported by recent EPR experiments on Glt Ph , which showed that the transport domain is structurally heterogeneous both in the apo and in the substratebound transporter, indicating a movement across the membrane in both conditions 19,20 .…”

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13,(26)(27)(28)(29)(30)(31)(32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra-and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward-and outward-facing conformations.…”