Local and global structure of the monomeric subunit of the potassium channel KcsA probed by NMR

Abstract

KcsA is a homotetrameric 68-kDa membrane-associated potassium channel which selectively gates the flux of potassium ions across the membrane. The channel is known to undergo a pH-dependent open-to-closed transition. Here we describe an NMR study of the monomeric subunit of the channel (KcsAM), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone 15N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in the gating of the tetrameric KcsA channel. An RDC-based model of KcsAM at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.

Figure 1. Secondary structure in KcsA^M vs KcsA^TET

Backbone chemical shifts of KcsA^M were measured to determine secondary structure elements of the channel subunit and contrasted with the homotetrameric channel assembly. Secondary ¹³C^α shifts (corrected for ²H isotope effects) of (top) 0.6 mM [²H,¹³C,¹⁵N]-labeled KcsA^M and (middle) 0.8 mM (monomeric concentration) [²H,¹³C,¹⁵N]-labeled KcsA^TET , both measured in 200 mM SDS and 25 mM MES pH 6.0 at 600 MHz and 323 K. Similar behavior is observed for the ¹³C′ secondary chemical shifts. (Bottom) Shown is a comparison of ¹H and ¹⁵N chemical shifts between the above samples of KcsA^M and KcsA^TET along the KcsA sequence. Δ_NH is defined as sqrt[(Δδ_H)² + (Δδ_N/5)²], with Δδ_H and Δδ_N representing the respective changes in ¹H and ¹⁵N chemical shifts, and the uncertainty in η_NH is estimated to be ≤ 0.02 ppm. For non-proline residues, missing bars indicate unassigned residues for one of the KcsA forms. Note that Δ_NH for residues 121–160 is ≤ 0.1 ppm. The cartoon above the figure delineates the positions of secondary structure elements as found in the KcsA crystal structure [2] and a previous NMR study [25].

Figure 2. pH-induced changes in KcsA^M secondary structure

(A) Secondary ¹³C^α shifts (corrected for ²H isotope effects) measured at 600 MHz and 323 K of [²H,¹³C,¹⁵N]-labeled KcsA^M in 200 mM SDS and (top) 25 mM sodium formate pH 4.2, and (middle) 20 mM Tris pH 8.0. Similar behavior is observed for the ¹³C′ secondary chemical shifts. (Bottom) Comparison of ¹H and ¹⁵N chemical shifts between KcsA^M samples at pH values of 4.2 and 8.0, with Δ_NH defined as in Figure 1. For clarity, bars are shown only for residues with Δ_NH ≥ 0.1 ppm. The cartoon above the figure delineates the positions of secondary structure elements as found in the KcsA crystal structure [2] and a previous NMR study [25]. (B) CD spectra of 15 μM samples of KcsA^M in 15 mM SDS at various pH values. Curves obtained in 25 mM sodium formate pH 4.2, 25 mM MES pH 6.0, and 20 mM Tris pH 8.0 are shown in red, blue (practically overlapping) and green, respectively. Predicted helical contents are ~51% of residues for pH 4.2 and 6.0, compared to only 45% for pH 8.0.

Figure 3. Solvent exchange of backbone ¹H^N protons in KcsA^M

Rates of exchange with solvent for KcsA^M backbone amide protons were estimated by lyophilizing a 0.5 mM KcsA^M sample in 200 mM SDS and 25 mM formate buffer, pH 4.2, and reconstituting in 99.9% D₂O. Shown is a [¹H,¹⁵N] tr-HSQC spectrum recorded 1 h after the sample was placed in a 600 MHz spectrometer at 323 K for the D2O sample. The total experiment time was 2 h. Visible peaks are labeled by residue number, with boxed labels showing no significant (<5%) change when compared to an identical spectrum recorded 7 hours later, and regular labels indicating peaks which were not visible or appreciably weakened in the second spectrum. Based upon these results, exchange rates at 323 K and pH 4.2 can be estimated as k_ex≤ 10⁻⁶ s⁻¹ for the former and 10⁻⁶ ≤ k_ex≤ 10⁻⁴ s⁻¹ for the latter.

Figure 4. ¹⁵N backbone relaxation for KcsA^M

¹⁵N backbone relaxation rates were measured at 600 MHz and 323 K for a 0.5 mM sample of [²H,¹³C,¹⁵N]-labeled KcsA^M in 200 mM SDS and 25 mM MES pH 6.0. Spin-locked transverse relaxation R₂ ^* values were derived from a measurement of R_1ρ against a spin-lock field of 1.8 kHz with an appropriate correction for offset effects [35]. Relaxation rates for residues 36–40 in the TM1 helix were not measured for this sample due to insufficient back-exchange of solvent protons at these sample conditions. Measurements repeated at pH 8.0 (data not shown) establish that relaxation rates are uniform throughout the TM1 helix. The cartoon above the figure delineates the positions of secondary structure elements as found in the KcsA crystal structure [2] and a previous NMR study [25].

Figure 5. Conformational exchange on the intermediate time scale in the TM2 domain

(A) Intensity of tr-HNCO peaks (arbitrary units) along the KcsA^M sequence. Spectrum was recorded at 600 MHz and 323 K for a 0.5 mM [²H,¹³C,¹⁵N]-labeled KcsA^M in 200 mM SDS and 25 mM formate buffer at pH 4.2. The cartoon above the figure delineates the positions of secondary structure elements as found in the KcsA crystal structure [2] and a previous NMR study [25]. (B) A comparison of ¹H, ¹⁵N-cross-correlated (η_xy) and transverse (R₂^*) relaxation rates (as described in Figure 4) for micelle-embedded residues at 600 MHz and 323 K for (left) a 0.5 mM sample of [²H,¹³C,¹⁵N]-labeled KcsA^M in 200 mM SDS and 25 mM MES pH 6.0, and (right) a similar sample containing 0.6 mM of KcsA^TET. In the interest of clarity, typical errors in both parameters are shown at the bottom right corner of each diagram. The two lines indicate the upper and lower limits of the expected scatter of η_xy vs. R₂ value assuming an average value of −170 ppm and a spread of ±30 ppm for the ¹⁵N chemical shift anisotropy. Values obtained for detectable residues in the TM2 helix segment ⁹⁸AGITSFGLV¹⁰⁷T are shown in red. Elevated R₂/ η_xy ratios (points above the upper line) are characteristic of an exchange contribution to transverse relaxation. Note that KcsA^TETη_xy and R₂ values exhibit a wider spread due to the rotational diffusion anisotropy of the KcsA-loaded micelle and the inclusion of amide sites from the PORE helix, which is the least collinear with the four-fold symmetry axis.

Figure 6. Measurement of D_NH residual dipolar couplings for KcsA^M

D_NH couplings were measured for a sample aligned in charged polyacrylamide gel (see text) containing 0.5 mM [²H,¹³C,¹⁵N]-labeled KcsA^M in 200 mM SDS and 25 mM formate buffer at pH 4.2, acquired at 600 MHz and 323 K. The downfield (blue) and upfield (magenta) resonances of the ¹H-coupled backbone ¹⁵N nuclei were obtained from an interleaved 3D tr-HNCO-based experiment as previously described [36]. Shown is a ¹H,¹⁵N plane obtained for a ¹³CO frequency of 178.5 ppm, demonstrating the importance of ¹³CO-separation for obtaining well-resolved couplings. The |¹ J_NH+ ¹ D_NH| splittings of selected backbone ¹H-¹⁵N sites are indicated, showing the opposite behavior of the TM1 (residues A32 and A42) and C-TER (residue E134) domains.

Figure 7. RDC-based models for the structure of SDS-solubilized KcsA^M

Shown are the sixteen different models for the three TM helical segments of KcsA^M, TM1 (blue), TM2^N (red), and TM2^C (yellow), which are compatible with the acquired RDC data. The TM1 helix is almost co-linear with the z-axis of the alignment frame, and its coordinates are maintained constant in all models. For the upper left structure, the positions of C^α atoms for residues L49, G88, V97, and L105 at the inter-helical ‘joints’ are shown as blue, red, orange and yellow spheres, respectively. Since the data provides no constraints on the translational relation between the three helical fragments, the models were created as follows: 1) The short six-residue linker (TM2^K) which connects the two TM2 fragments was appended to each of them in a helical conformation, and the two virtual T101 C^α atoms were superimposed; 2) Since TM1 and TM2^N are linked by a solvent-exposed helical segment, residues L49 and G88 have been located on the same side of the SDS micelle. The G88 C^α atom was placed in a plane perpendicular to TM1 and containing the L49 C^α atom, and 7 Å away from the TM1 helical axis. While the position of TM2 is still ill-defined in this procedure, the models shown aim to minimize the radius of gyration and create a relatively compact structure compatible with backbone ¹⁵N relaxation and light-scattering data. Of the sixteen possibilities, structures F and G provide the most compact arrangement of the helices, and the former is depicted in the cartoon in Figure 8.

Figure 8. Model of SDS-solubilized KcsA^M

RDCs measured for the micelle-embedded helices of KcsA^M were used to model this domain. (A) All 90 RDCs (normalized to D_NH values) fitted to the structure of the monomeric subunit within the KcsA^TET structure (upper cartoon). (B) Same as (A), except the TM2 helix was allowed to bend at the kinked segment to optimize the fit between experimental and calculated RDC values, leading to a reduction in Q-factor from 0.67 to 0.35. The resulting model is shown (lower cartoon). (C) The RDC-based model of KcsA^M embedded in an SDS micelle and aligned in a prolate polyacrylamide gel cavity.