Pentameric assembly of the Kv2.1 tetramerization domain

Figures

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  Figure 1. Overview of the Kv2.1 T1 crystal structure. (a, b) Cartoon representation of (a) the top view (C-terminus) and (b) the side view of the X-ray structure of Kv2.1 T1 in a pentameric form (PDB entry 7re5). (c, d) Top view of previously reported X-ray structures of (c) the pentamer of KCTD5 (PDB entry 3drz) and (d) the tetramer of Kv4.2 (PDB entry 1nn7). Colors for secondary structure are α-helices in cyan and β-sheets in magenta; the gray sphere is the zinc ion.

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  Figure 2. The monomer–monomer interface of Kv2.1 T1 is a conserved charged surface. (a) Surface representation of Kv2.1 T1 colored by electrostatic potential calculated using APBS. The gradient is from −5kT/e (red) to +5kT/e (blue). Top: as observed from the side view of the X-ray structure in Fig. 1 ▸(b). Bottom: subunits are rotated 90° with respect to the X-ray structure. (b) Surface representation of Kv2.1 T1 colored by sequence conservation (purple for most conserved and green for least conserved). Conservation scores were calculated using ConSurf. The top and bottom views are subunits rotated as described for (a).

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  Figure 3. Pentamer formation involves inter-subunit zinc binding and a salt bridge. (a) Structure-based multiple sequence alignment of Kv2.1 with KvS (Kv9.3, Kv8.2 and Kv6.4), Kv3.1, Kv4.1 and Kv1.1 T1 domains. Blue triangles below residues indicate the Zn2+-coordination motif HX 5CX 20CC and magenta circles indicate the negatively charged CDD motif and its interacting positive residues at the N-terminus. Shading of residues in blue or magenta, respectively, highlights conservation. (b) Cartoon representation of Kv2.1 T1 with residues at the subunit interface highlighted as sticks; colors are chain A in green and chain B in orange. The insets show the interacting residues in the Zn2+-coordination site (left) or CDD motif (right). Hydrogen bonds are shown as black dashed lines, the zinc ion is in gray, O atoms are in red, S atoms are in yellow and N atoms are in dark blue.

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  Figure 4. MALS–SAXS analysis of Kv2.1 T1. (a) MALS analysis of Kv2.1 T1. The calculated molecular weight (mean ± SD) is taken from the portion of the light-scattering peak indicated in red. (b) The experimental scattering curve of Kv2.1 T1 (black) is compared with theoretical scattering curves generated by CRYSOL for a tetrameric homology model of Kv2.1 T1 generated using YASARA based on Kv3.1 T1 (PDB entry 3kvt, blue, χ2 = 27) and the pentameric Kv2.1 T1 crystal structure (green, χ2 = 1.7). (c) The ab initio model of Kv2.1 T1 (shown as a surface) was reconstructed using GASBOR and the averaged filtered shape from DAMFILT is shown. The model is superimposed with cartoons for the tetrameric Kv2.1 T1 YASARA homology model (left) and the pentameric Kv2.1 T1 crystal structure (PDB entry 7re5; right).

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  Figure 5. Negative-stain transmission electron microscopy of Kv2.1 T1. (a) An electron micrograph of a Kv2.1 T1 sample negatively stained with 2%(w/v) uranyl acetate and imaged at 100 000× magnification. Inset: increased magnification to aid the visualization of individual oligomers; two examples are outlined with yellow dashed lines. (b) 2D reference-free representative class averages of particles in a top view revealed a pentameric form for Kv2.1 T1.

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  Figure 6. Protein thermal unfolding in the presence of EDTA. (a) Comparison of CD spectra of Kv2.1 T1 at 10°C in the absence (black) or presence (red) of 1 mM EDTA with the calculated spectra for Kv1.1 (PDB entry 1t1d, purple), Kv3.1 (PDB entry 3kvt, blue) and Kv4.3 (PDB entry 1s1g, light blue) T1 domains. (b) Stability analysis of Kv2.1 T1 in solution using CD. The fraction of unfolding extracted from far-UV CD spectra at 222 nm was plotted as a function of temperature. (c) The hydrodynamic radius of Kv2.1 T1 monitored by dynamic light scattering is presented as a function of temperature. In (b) and (c), the black data points are for Kv2.1 T1 without EDTA and the red data points are for Kv2.1 T1 treated with 1 mM EDTA.