Homology models of the tetramerization domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6

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1 Homology models of the tetramerization domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6 Hsuan-Liang Liu* and Chin-Wen Chen Department of Chemical Engineering and Graduate Institute of Biotechnology, National Taipei University of Technology Abstract The homology models of the tetramerization (T1) domain of six eukaryotic potassium channels, Kv1.1- Kv1.6, were constructed based on the crystallographic structure of the Shaker T1 domain. The results of amino acid sequence alignment indicate that the T1 domains of these K + channels are highly conserved, with the similarities varying from 77 % between Shaker and Kv1.6 to 93 % between Kv1.2 and Kv1.3. The homology models reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K + channel. These models also show that each T1 monomer consists of three distinct layers, with N-terminal layer 1 and C-terminal layer 3 facing the cytoplasm and the membrane, respectively. Layer 2 exhibits the highest structural conservation because it is located around the central hydrophobic core. For each Kv channel, four identical subunits assemble into the homotetramer architecture around a four-fold axis through the hydrogen bonds and salt bridges formed by 15 highly conserved polar residues. The narrowest opening of the pore is formed by the four conserved residues corresponding to R115 of the Shaker T1 domain. The homology models of these Kv T1 domains provide particularly attractive targets for further structure- based studies. Keywords: Tetramerization (T1) domain, Potassium channel, Amino acid sequence alignment, Homology, Hydrogen bond, Salt bridge. Introduction Potassium channels are now known in virtually all types of cells in all organisms, where they are involved in multitude of physiological functions (1). Voltage-gated K + channels are multisubunit ion channels with multidomains. Among them, the Shaker K + channel is the most thoroughly studied one, and extensive mutagenesis has been carried out to gain insight into its structure and function relationships (2) Amino acid sequence analysis of the Shaker gene predicts six hydrophobic and α-helical transmembrane (TM) segments termed S1-S6, where S1-S4 form the voltage-sensor domain and S5 and S6 along with the pore helix constitute the pore loop domain. However, in the Shaker K + channel, only ~30% of the protein resides within the membrane, and this is franked by two cytoplasmic segments: the ~220-residue N-terminus and the ~150-residue C-terminus (3) The N-terminal region includes a so-called tetramerization (T1) domain. It has been shown that Shaker K + channels remain functional when most of the N-terminal

2 domain is removed (4). Recently, the X-ray crystallographic structures of pore loop domain of the bacterial K + channel from Streptomyces lividans (KcsA) (5) and of the voltage-gated K + channel from Aeropyrum pernix (KvAP) (6) have been determined. The structural features of the pore loop domain shows for the first time how ion selectivity can be realized by tetrameric coordination of ions in the channel pore (5). Furthermore, the newly solved KvAP structure (6) has allowed the principle of gating charge movement, which is associated with channel opening, to be elucidated (7). On the other hand, the atomic structure of the T1 domain of the Shaker K + channel has been also solved. The structure of the T1 monomer consists of three distinct layers (8). The T1 domain forms a tetramer either in solution (8) or in the fully assembled channel (3). The four identical subunits are arranged in a four-fold symmetry surrounding a centrally located pore about 20 Å in length (8). A recent crystal structure of the entire cytoplasmic β-subunit also indicates a fourfold symmetric complex. It has been suggested that in order to provide access to the pore, T1 must be separate from the membrane domain similar to a hanging gondola. A three-dimensional (3D) structure of the entire Shaker channel at 2.5 nm resolution has provided further evidence for the hanging gondola model. Although the precise relationship between the TM and T1 domains in a functional Kv channel is still unknown, both domains form rotationally symmetric tetrameric structures, suggesting that they both lie at the central axis of the Kv channel (9). Materials and Methods Structural homology to construct the structural models of the T1 domains of the Kv1.1-Kv1.6 K + channels was based on the crystal structure of the Shaker T1 domain (8) obtained from the protein data bank (PDB; accession number 1T1D). Unfavorable nonphysical contacts in this structure were eliminated using Biopolymer module of Insight II program (Accelrys, San Diego, CA, USA) with the force field Discover CVFF (consistent valence force field)(10) in the SGI O200 workstation with 64-bit HIPS RISC R MHz CPU and PMC-Sierra RM7000A 350MHz processor (Silicon Graphics, Inc., Mountain View, CA, USA), followed by 10,000 energy minimization calculations using steepest descent method, to yield the model protein for further structure building. The force field parameters in CVFF were developed by computing the properties of nearly 2000 different macromolecules such as proteins, nucleic acids, carbohydrates, and lipids, resulting in over two million quantum mechanically computed energies and energy derivatives(10). Homology utilizes structure and sequence similarities for predicting unknown protein structures (Fig 1). The Homology module in Insight II allows us to build a 3D model of the target protein (i.e., the T1 domain of Kv1.1-Kv1.6) using both its amino acid sequence and the structures of known, related model proteins (i.e., Shaker T1 domain).

3 Results and Discussion Point, insertion, or deletion mutations that would result in a critical loss of biological functions are less favored by evolution and consequently, functionally and structurally relevant domains tend to be highly conserved across a corresponding protein family. Such conservation can be detected as a pattern of conserved residues. Therefore, an optimal amino acid sequence alignment based on the conserved residues is essential to the success of structural homology. The results of pairwise amino acid sequence alignment of the T1 domains of Kv1.1-Kv1.6 to that of Shaker K + channel are given in Fig. 1. The amino acid sequences in the T1 domains of these K + channels are highly conserved, with the similarities varying from 77 % between Shaker and Kv1.6 to 93 % between Kv1.2 and Kv1.3. Previously, multiple sequence alignment of the T1 domains from various Kv K + channels has identified 24 highly conserved residues including seven absolute identities (N71, G74, L92, F111, L122, Y125, and G128). The present alignment also identifies these highly conserved residues among Shaker and Kv1.1-Kv1.6 K + channels. As mentioned previously, most of these highly conserved residues are located in the hydrophobic core of the T1 monomer, the overall fold of the T1 domain is well preserved among various K + channels. Thus, it is reasonable to construct the homology models of the Kv1.1-Kv1.6 T1 domains based on the crystallographic structure of the Shaker T1 domain After energy minimization calculations, the structure of the Shaker T1 monomer consists of three distinct layers: 1) The N-terminal layer 1 (residues ) is formed by two pairs of antiparallel β-strands (β1-β2 and β3-β4) interrupted by two α-helices (α1 and α2); 2) Layer 2 (residues ) is a 12-residue-long segment including α3; and 3) The C-terminal layer 3 (residue ) consists of two α-helices (α4 and α5) (see Fig 2). Residues in layer2 are more conserved than those in layers 1 and 3 among these K + channels, indicating that the structure of layer 2 is more preserved among these K + channels. Interestingly, layer 3 consists of a single β-strand and an α-helix in the crystallographic structure of Shaker T1 domain, whereas the single β-strand in layer 3 has been replaced by an α-helix as predicted by DSSP in the present study. Despite this difference, other secondary structures predicted by DSSP are very similar to those defined in the crystallographic structure, indicating that not only the amino acid sequences but also the secondary structures are highly conserved in the T1 domains of Shaker and Kv1.1-Kv1.6 K + channels. In addition, fourteen out of 15 residues involved in polar intersubunit interactions (E78, T79, Q80, T83, D119, Q126, and R130 from subunit 1 and N71*, S73*, R76*, D112*, R115*, D140*, and E144* from subunit 2) are totally conserved, despite that residue R132 is replaced by Lys for Kv1.4. The results suggest that T1 domain also undergoes extensive RNA editing at very highly conserved interface positions, which can profoundly alter expression levels and tetrameric assembly. Additional diversity of the Kv channels can

4 probably be created through additional gene splicing. Furthermore, Kv channel diversity is expanded by the homo- and heterotetrameric assembly of the subunits into functional channels. The inherent K + channel diversity and the regulation of channel formation presumably provide a means for diversification of K + channel properties so that cells can adapt various gating and assembling mechanisms. In summary, using amino acid sequence alignment and structural homology, a set of homology models of the T1 domain of the eukaryotic voltage-dependent K + channels, Kv1.1 -Kv1.6 (Fig 2), were generated based on the crystallographic structure of the Shaker T1 domain. These homology models show that the T1 domain of these Kv channels shares the similar folds as in the Shaker T1 domain. In particular, the structural feature and the specific packing of the three distinct layers are similar to those of Shaker, with layer 2 exhibits the highest structural conservation (Fig 3). The intersubunit interactions governing the assembling of these four subunits for the Shaker and Kv1.1-Kv1.6 T1 domains are mediated by 14 totally and 1 highly conserved residues. The total volume, surface area, and pore size of the Kv1.1-Kv1.6 T1 tetramers are similar to those of Shaker T1 tetramer (Fig 4), strongly suggesting that these T1 domains share a common overall structural architecture. Although these are only theoretical models, they still provide particularly attractive targets for structure-based studies, such as investigating the assembling process of these T1 domains through MD simulations, which are currently conducted in our laboratory. Acknowledgements Financial support from National Science Council of Taiwan is highly appreciated (NSC E ). References (1) Biggin, P. C.; Roosild, T.; Choe, S. Curr. Opin. Struct. Biol. 2000, 10, (2) Sigworth, F. J.. Rev. Biophys. 1994, 27, (3) Kobertz, W. R.; Williams, C.; Miller,C. Biochemistry 2000, 39, (4) Tu, L. W.; Stantarelli, V.; Deutsch, C. Biophys. J. 1995, 68, (5) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. Science 1998, 280, (6) Jiang, Y.; Lee, A.; Chen, J.; Ruta, V.; Cadene, M.; Chait, B. T.; MacKinnon, R. Nature 2003, 423, (7) Jiang, Y.; Ruta, V.; Chen, J.; Lee, A.; MacKinnon, R. Nature 2003, 423, (8) Kreusch, A.; Pfaffinger, P. J.; Stevens, C. F.; Choe, S. Nature 1998, 392, (9) Cushman, S. J.; Nanao, M. H.; Jahng, A. W.; DeRubeis, D.; Choe, S.; Pfaffinger, P. J. Nature Struct. Biol. 2000, 7, (10) Kabsch, W.; Sander, C. Biopolymers 1983, 22,

5 Fig.1 Amino acid sequence alignment of the Shaker and Kv1.1-Kv1.6 T1 domains. The locations of secondary structures predicted by DSSP (10) are shown. The start and end amino acid residues are numbered in the brackets on the left and right of each sequence, respectively. Residues totally conserved in all sequences are indicated in red letters with green background. Residues conserved in the Kv family but different from those in Shaker are represented in blue letters with indigo background. Residues where variations occur are given in black letters with yellow background. The totally conserved residues identified from the previous multiple sequence alignment (8) are indicated as closed square symbols. The residues involved in polar intersubunit interactions are indicated as asterisk symbols. Fig.2 Ribbon (left) and molecular surface representations (right) of the T1 monomer of (A) Shaker (B) Kv1.4. The structural features corresponded to those defined in the crystallographic structure of the Shaker T1 domain (8) are indicated. The polypeptide backbones belonging to the α-helix, β-strand, turn and random coil regions are shown in red, yellow, blue and green, respectively. The backbone RMSDs of Kv1.4 after superimposing the homology models to the crystallographic structure of Shaker are given on the top right corner of each model. The residues involved in polar intersubunit interactions are shown in red in each molecular surface model.

6 Fig. 3 The cross-sectional views of (A) layer 1; (B) layer 2; and (C) layer 3 after superimposing the backbone atoms of the Kv1.1-Kv1.6 T1 tetramers to the Shaker T1 tetramer. These structures are viewed from the cytoplasmic side. Fig.4 Molecular surface representations of the top (upper) and side (lower) views of the T1 tetramer of Kv1.1. The residues involved in polar intersubunit interactions are labeled. The top view of the T1 tetramer is viewed from the membrane side. One of the four subunits of these channels is omitted in the side view for clarity. The narrowest opening in the center of the pore is formed by the conserved residues corresponding to R115 in the Shaker T1 domain.