Crystal structure of full-length KcsA in its closed conformation

Uysal, Serdar; Vásquez, Valeria; Tereshko, Valentina; Esaki, Kaori; Fellouse, Frédéric A.; Sidhu, Sachdev S.; Koide, Shohei; Perozo, Eduardo; Kossiakoff, Anthony A.

doi:10.1073/pnas.0810663106

Cited by 216 publications

(241 citation statements)

References 36 publications

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“…1, dashed lines). The overall channel architecture was in good agreement with the WT KcsA crystal structure (25). In detail, however, we observed significant differences with respect to the TM1 (S5) pore-loop region.…”

Section: Resultssupporting

confidence: 81%

“…We used an ssNMR-based hybrid strategy to establish 3D structural views of Chim before and after C-type inactivation. First, we constructed a homology model of the closed conductive state by using the crystal structure of KcsA (25) [Protein Data Bank (PDB) ID code 3EFF] and constructed a tetramer by using high ambiguity driven docking (HADDOCK) (26) (SI Methods). This model then served to predict and evaluate experimental CC and CHHC correlation experiments as the basis of 3D molecular structures (e.g., ref.…”

Section: Resultsmentioning

confidence: 99%

“…Potassium and chloride ions were added to electrostatically neutralize the systems and to mimic 80 mM KCl solutions. The KcsA starting structure was derived from crystal structure 3EFF (PDB) (25) to probe spontaneous helical elongation of TM1, the Chim starting structure from our ssNMR-based structure model. Mutations in the Chim model were introduced with Pymol (Delano Scientific).…”

Section: Methodsmentioning

confidence: 99%

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Importance of lipid–pore loop interface for potassium channel structure and function

Cruijsen

Nand

Weingarth

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Potassium (i.e., K + ) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K + channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K + channel inactivation and exhibits a remarkable structural plasticity that correlates to K + channel inactivation. The transmembrane helix 1 unwinds when the K + channel enters the inactivated state and rewinds during the transition to the closed state. In addition to wellcharacterized changes at the K + ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K + channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K + channel pore. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Importance of lipid–pore loop interface for potassium channel structure and function

Cruijsen

Nand

Weingarth

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The crystals from the two double mutants diffracted to 3.8 Å (L9′S + F16′L) and 4.2 Å (L9′A + F16′L), a resolution that, albeit low, is comparable to that of other X-ray crystal structures of agonist-bound (10) and full-length (29,30) bacterial channels. Evidently, this resolution is too low to allow the determination of the conformation of individual side chains or to detect the rearrangement of loop regions unambiguously, but it is enough to detect large changes in the orientation of the transmembrane α-helices of the kind that would make the pore of ELIC look like that of GLIC.…”

Section: Resultsmentioning

confidence: 56%

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

González-Gutiérrez

Lukk

Agarwal

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

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“…Additional difficulties, such as conformational heterogeneity and inherent protein flexibility can be reduced by inhibitors or antibodies rigidifying the structure of the target protein. These methodological advances have contributed to the crystallization of a broad range of membrane-bound signalling proteins (Rasmussen et al, 2011a;Uysal et al, 2009;Krishnamurthy and Gouaux, 2012).…”

Section: Introductionmentioning

confidence: 99%

Asymmetric perturbations of signalling oligomers

Maksay

Tőke

2014

Progress in Biophysics and Molecular Biology

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This review focuses on rapid and reversible noncovalent interactions for symmetric oligomers of tetramers (voltage-gated cation channels, ionotropic glutamate receptor, CNG and CHN channels); pentameric ligand-gated and mechanosensitive channels; higher order oligomers (gap junction channel, chaperonins, proteasome, virus capsid); as well as primary and secondary transporters. In conclusion, asymmetric perturbations seem to play important functional roles in a broad range of communicating networks.

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Crystal structure of full-length KcsA in its closed conformation

Cited by 216 publications

References 36 publications

Importance of lipid–pore loop interface for potassium channel structure and function

Importance of lipid–pore loop interface for potassium channel structure and function

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

Asymmetric perturbations of signalling oligomers

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