Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux

Hänelt, Inga; Wunnicke, Dorith; Müller-Trimbusch, Meike; Brüggen, Marc Vor der; Kraus, Inga; Bakker, Evert P.; Steinhoff, Heinz‐Jürgen

doi:10.1074/jbc.m110.139311

Cited by 30 publications

(34 citation statements)

References 49 publications

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“…The four-pulse DEER sequence was applied 21 . All parameters were kept unmodified with respect to previously described values 22 , with observer pulses of 16-32 ns and a pump pulse of 12 ns. Sample volumes of 30-40 µl were loaded into EPR quartz tubes (3 mm outer diameter) and shock frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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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: Methodsmentioning

confidence: 99%

Conformational heterogeneity of the aspartate transporter GltPh

Hänelt

Wunnicke

Bordignon

et al. 2013

Nat Struct Mol Biol

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104

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Section: Ion Channelsmentioning

confidence: 99%

“…Besides nowadays various structures a plethora of EPR studies has been performed with regard to conformational changes in the selectivity filter [65,66], the cytoplasmic helical bundle [67,68] and the communication between both features [69]. Further excellent examples for the combined use of structural biology and EPR spectroscopy are present for all classes of ion channels, e.g., for the intracellular Mg 2+ -gated Mg 2+ channel CorA [70][71][72], the nucleotide-and Na + -gated K + channel KtrAB [73], the mechanosensitive channel MscL [74,75], the cyclic AMP-gated HCN channel [45,76], the voltage-gated Na + channel NavMs [77] and the pentameric receptor channels GLIC [78]. In all cases EPR measurements have been applied to validate existing protein structures, determine the structure of missing regions or distorted elements in structures, or add valuable information of the dynamics of the studied systems to provide a model of activation and regulation.…”

Section: Ion Channelsmentioning

confidence: 99%

“…A particular challenge for the investigation of ion channels by EPR spectroscopy is their mostly homo-oligomeric nature, which requires the interpretation of various distance distributions even if only one spin label is inserted per protomer [80]. A great advantage over most structural studies is the possibility to perform EPR measurements on liposome-reconstituted proteins such that electrochemical gradients as needed for the open conformation can be applied [73].…”

Section: Ion Channelsmentioning

confidence: 99%

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The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

Wunnicke

Hänelt

2017

Crystals

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Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.

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“…An open-source software (DEER Analysis 2011) for extracting distance distributions from DEER data sets has been provided by the ETHZurich (http://www.epr.ethz.ch/software/index). DEER gave a deeper inside the transmembrane signaling mechanism of rhodopsin (Altenbach et al 2008;Knierim et al 2008), sensory rhodopsin (Holterhues et al 2011;Klare et al 2011), the maltose ABC transporter (Grote et al 2008;Grote et al 2009) and the KtrAB potassium transporter (Hänelt et al 2010). Thus, during recent years and on the basis of crystallographic data, DEER has been established as the state of the art technique to allow description of signal and transport mechanisms (Bordignon, Grote, and Schneider 2010;Klare and Steinhoff 2010).…”

Section: Site-directed Spin Labeling Electron Paramagnetic Resonancementioning

confidence: 99%