Structural perspectives on secondary active transporters

Boudker, Olga; Verdon, Grégory

doi:10.1016/j.tips.2010.06.004

Cited by 146 publications

(134 citation statements)

References 85 publications

(109 reference statements)

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“…These observations suggest that the equilibrium constants between the observed transporter conformations are close to unity and, consequently, that different states are populated to similar extents. This observation makes sense for the empty carrier and for Glt Ph bound to both Na + and aspartate 17 , as these forms need to be able to isomerize between outward-and inward-facing states for substrate binding and release. However, the Na + -bound protein would not be expected to alternately expose the binding site, as this would lead to uncoupled Na + leaks.…”

Section: Discussionmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

104

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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...

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Section: Discussionmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

104

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“…SITs possess an inverted symmetrical structure of 5 þ 5 TMDs ( figure 4). This is characteristic of the LeuT-fold-type sodium symporters, which all share a common inverted 5 þ 5 TMD structural psuedosymmetry [45,46]. The degree of symmetry observable from the amino acid sequence of SITs is notable in comparison with other LeuT-fold transporters, whose inverted pseudosymmetry is only discernible from their three-dimensional structure and is not visible in the primary protein sequence [45].…”

Section: (D) Proposed Structure and Function Of Choanoflagellate Silimentioning

confidence: 98%

A family of diatom-like silicon transporters in the siliceous loricate choanoflagellates

et al. 2013

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Biosilicification is widespread across the eukaryotes and requires concentration of silicon in intracellular vesicles. Knowledge of the molecular mechanisms underlying this process remains limited, with unrelated silicontransporting proteins found in the eukaryotic clades previously studied. Here, we report the identification of silicon transporter (SIT)-type genes from the siliceous loricate choanoflagellates Stephanoeca diplocostata and Diaphanoeca grandis. Until now, the SIT gene family has been identified only in diatoms and other siliceous stramenopiles, which are distantly related to choanoflagellates among the eukaryotes. This is the first evidence of similarity between SITs from different eukaryotic supergroups. Phylogenetic analysis indicates that choanoflagellate and stramenopile SITs form distinct monophyletic groups. The absence of putative SIT genes in any other eukaryotic groups, including non-siliceous choanoflagellates, leads us to propose that SIT genes underwent a lateral gene transfer event between stramenopiles and loricate choanoflagellates. We suggest that the incorporation of a foreign SIT gene into the stramenopile or choanoflagellate genome resulted in a major metabolic change: the acquisition of biomineralized silica structures. This hypothesis implies that biosilicification has evolved multiple times independently in the eukaryotes, and paves the way for a better understanding of the biochemical basis of silicon transport through identification of conserved sequence motifs.

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“…This 'alternating access' model was proposed almost half a century ago, long before structures of membrane transporters were solved 40,41 , and applies to all membrane transporters, not just to the ABC transporter family. The many crystal structures that are now available for different transport proteins provide insights into the molecular mechanisms that mediate alternating access (recently reviewed in REFS 28,42,43). Three models for transport have been proposed -the rocker-switch mechanism, the gated-pore mechanism and the elevator mechanism (FIG.…”

Section: Transport Mechanismmentioning

confidence: 99%

“…3c) along which the transport domain can move. The elevator mechanism is considered to be a manifestation of the 'moving-carrier mechanism' [42][43][44][45][46] .…”

Section: Transport Mechanismmentioning

confidence: 99%