Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

Takeuchi, Koh; Takahashi, Hideo; Kawano, Seiko; Shimada, Ichio

doi:10.1074/jbc.m608264200

Cited by 78 publications

(111 citation statements)

References 36 publications

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“…TFE is also known to increase the pK a values of p-or o-hydroxybenzoic acids (28). Because the activation of KcsA is triggered by the protonation of H25 near the helix bundle crossing (10,15), changes in the pK a values of the acidic residues by the addition of TFE can potentially modulate the structural equilibrium of KcsA. However, the increase in pK a by 25% TFE is only 0.5 points for hydroxybenzoic acids, and the effect would be smaller in the TFE concentration range used here.…”

Section: Discussionmentioning

confidence: 99%

Functional Equilibrium of the KcsA Structure Revealed by NMR

Imai

Osawa

Mita

et al. 2012

Journal of Biological Chemistry

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Background:The selectivity filter of KcsA undergoes an equilibrium between permeable and impermeable conformations under acidic conditions. Results: Truncation of the intracellular region or addition of 2,2,2-trifluoroethanol modulates the equilibrium. Conclusion: Membrane environments affect dynamics of KcsA. Significance: This is the first evidence that a structural equilibrium in the membrane is related to the inactivation of a potassium channel.

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Section: Discussionmentioning

confidence: 99%

Functional Equilibrium of the KcsA Structure Revealed by NMR

Imai

Osawa

Mita

et al. 2012

Journal of Biological Chemistry

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“…Given the overall similarity in folding, it is tempting to presume that the mechanism of pore opening probability is properly represented in the WSK3 construct. Recently, H25 (H4 in WSK3) was reported to act as the pH sensor in opening the hydrophobic gate at the cytoplasmic side of KcsA (22). H25 lies in a hydrophobic intersubunit region in both the WSK3 and KcsA structures.…”

Section: Discussionmentioning

confidence: 99%

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Ma¹,

Tillman²,

Tang³

et al. 2008

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

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Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics. membrane protein ͉ protein design ͉ potassium channels ͉ slow inactivation L ess than 1% of known protein structures belong to transmembrane (TM) proteins. The scarcity of structural data is due to the difficulties associated with handling membrane proteins for structure determination. With few exceptions, membrane proteins are often present at low levels in natural tissues, and the available cellular machinery for membrane insertion often limits the capacity of high-level expression systems. Outside their native environment, membrane proteins are unpredictable in behavior, with proper folding and stability depending on the choice of the membrane mimetic environment in addition to the common variables affecting soluble proteins. Although some membrane proteins can be correctly refolded after misfolding or aggregation during expression and purification, others are affected by these processes irreversibly. Currently, no reliable method is available to predict which membrane mimetic will stabilize a given protein in its native conformation without aggregation, and often an arduous process of trial and error using expensive reagents must be carried out to find appropriate conditions.An intriguing alternative to inserting TM proteins into a membrane mimetic environment for structure determination is to alter the proteins such that a membrane is no longer required. Designing water-soluble analogues of membrane proteins challenges the basic understanding of what st...

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“…To explore the dynamics involved in channel conductance, we studied the conformational exchange dynamics of KcsA, also examined by others 19,26,27 , using NMR spectroscopy. Our variants are all closed at pH 7, but they differ in open probability and mean open time at pH 4 ( Table 1) Figure 2c and Figure 4 show titration measurements of Tyr78 from wild-type KcsA, KcsA(E71A), and KcsA(tox) with the R64D mutation mimicking the R64A variant.…”

Section: Conformational Dynamics Of Kcsa In the Open Statementioning

confidence: 99%

Conformational dynamics of the KcsA potassium channel governs gating properties

Baker

Tzitzilonis

Kwiatkowski

et al. 2007

Nat Struct Mol Biol

125

136

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K + channels conduct and regulate K + flux across the cell membrane. Several crystal structures and biophysical studies of tetrameric ion channels have revealed many of the structural details of ion selectivity and gating. A narrow pore lined with four arrays of carbonyl groups is responsible for ion selectivity, whereas a conformational change of the four inner transmembrane helices (TM2) is involved in gating. We used NMR to examine full-length KcsA, a prototypical K + channel, in its open, closed and intermediate states. These studies reveal that at least two conformational states exist both in the selectivity filter and near the C-terminal ends of the TM2 helices. In the ion-conducting open state, we observed rapid structural exchange between two conformations of the filter, presumably of low and high K + affinity, respectively. Such measurements of millisecond-timescale dynamics reveal the basis for simultaneous ion selection and gating.The simplest structural definition of ion channel gating is the ability to alternate, in a signaldependent way, between states that selectively permit the flow of ions (open) or do not (closed). In the closed state, generally a default state, K + channels inhibit ion flow with high efficacy. In the open state, two opposing tasks must be carried out near the diffusion limit: ion selection and permeation 1 . Energetically, ion selection requires spatial coordination that favors K + over other cations, whereas permeation requires the rapid release of the cation from these recognition sites. This 'hold-and-release' feature underlies the unique structural and dynamical properties of the channel.Crystal structures of K + channels of prokaryotic origin (KcsA 2 , MthK 3 , KirBac1.1 (ref. 4), KvAP 5 and NaK 6 ) have revealed a universal structural framework for these tasks. To explain ion selectivity, the structures show a narrow filter composed of four successive layers of main chain carbonyl oxygen atoms, which act as surrogate waters for the dehydrated K + ions (1.35-Å radius) as they pass through the filter. This unique spatial geometry results from the signature filter amino acid sequence, Gly-Tyr-Gly, in which the two glycines enable positive φ/ψ angles of the protein backbone so that the K + -coordinating carbonyl oxygens can all point inward to the pore axis simultaneously 7 to perform ion selection 8,9 . For gating of the channel, the pore-lining C-terminal ends of the inner AUTHOR CONTRIBUTIONSK.A.B. and C.T. prepared KcsA; K.A.B., C.T., W.K. and R.R. collected and analyzed NMR data; K.A.B., C.T., S.C. and R.R. contributed to scientific discussions and prepared the manuscript. Fig. 3 online), we have obtained B85% of the sequential backbone assignments of full-length KcsA(tox) at pH 7 (Fig. 1). A residue-specific secondary structure analysis based on positive 13 Cα chemical shift deviations from a random coil (Fig. 1a), amide-H 2 O exchange ( Supplementary Fig. 4b online) and protein-detergent NOEs ( Supplementary Fig. 4a) revealed the presence of the following he...

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Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

Cited by 78 publications

References 36 publications

Functional Equilibrium of the KcsA Structure Revealed by NMR

Functional Equilibrium of the KcsA Structure Revealed by NMR

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Conformational dynamics of the KcsA potassium channel governs gating properties

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