Crystal structure of the leucine zipper domain of small‐conductance Ca2+‐activated K+ (SKCa) channel from Rattus norvegicus
The leucine zipper (LZ) is a typical member of the coiled coil family, the most common and extensively investigated of structural motifs.1 In terms of their biological functions, LZs participate in a variety of cellular processes, supplying unique protein–protein interactions via leucine–leucine zipping. The LZ domain is defined as an α-helix comprised of heptad repeats (abcdefg)n in which the residues at positions a and d are hydrophobic and mediate critical interhelical interactions, while b, c, e, f, and g are hydrophilic and form the solvent-exposed part of the coiled coil.2-4 Moreover, two or more LZ domains can intertwine in parallel or antiparallel to form a bundle of α-helices that interact via hydrophobic interactions at the inner face of the coiled coils.5-7 LZ domains were originally identified as highly conserved motifs mediating the interaction between transcriptional factors, but are now known to also be present in a number of ion channels.8 The sequences of ion channel LZ domains contain features common to the transitional LZs of transcriptional factors,2, 4 but the canonical leucine at position d is often replaced by other residues such as isoluecine, valine, or even a nonhydrophobic residue, thereby forming a modified LZ.9 Recent reports on the function of ion channel LZ domains have suggested that they participate in regulating the channel's activity by targeting modulator proteins to the channel, promoting formation of "macromolecular signaling complexes."8-16 For instance, the LZ domains of the RyR2 channel recruit adaptor molecules for PKA, PP1, and PP2A, which regulate channel activity through phosphorylation of certain residues.9-11 They also facilitate correct folding and plasma membrane trafficking of ion channels.17-19 By substituting leucine residues, it was confirmed that "leucine– leucine zipping" between the LZs of ion channels and partner proteins is required for assembly of these signal complexes.11, 15, 17 For instance, the targeting of modulator molecules to the RyR2 channel was impaired when alanine was substituted for the leucine at position d of the LZ.10 In other words, ion channels and signaling complexes form specific LZ heteromers within the macromolecular signaling complexes.20 Thus LZ domains appear to be the major binding motif mediating the interaction between intracellular proteins involved in signal transduction and ion channels. Here we describe our analysis of the 2.1 Å X-ray crystal structure of the LZ domain of the small conductance voltage-independent Ca2+-activated K+ channel (SKCa) from rat (Rattus norvegicus). Intriguingly the structure of the LZ domain is characterized by a parallel trimeric α-helix bundle that is incompatible with the tetrameric state of the functional SKCa channel. The open reading frame of the LZ domain (aa. #488–#526) of the rat SKCa channel isoform 2 (U69882) was amplified by PCR using the following primers: 5′-GGAATATCATGTATGATATG-3′ (forward) and 5′-TTAAGGAGGGCATGAAT-3′ (backward). Chromosomal DNA encoding the rat SKCa channel served as the template for the PCR amplification. After digestion with NdeI and BamHI (NEB), the amplified DNA fragment was cloned into a modified pGEX vector (Pharmacia) so that the LZ domain would be expressed with glutathione S-transferase (GST) fused at its N-terminus for affinity purification. The resultant expression plasmid was confirmed by sequencing and used to transform E. coli strain BL21(DE3). The overexpressed LZ domain was initially purified using glutathione Sepharose 4B affinity resin (Peptron), after which the GST-fused proteins were eluted with 15 mL of elution buffer containing 20 mM reduced glutathione in 50 mM Tris–HCl (pH 8.0) and 0.2M NaCl. To remove the GST moiety, the eluted proteins were incubated in the presence of 40 U of thrombin with mild rotation. The digested LZ proteins including extra four residues (GSHM) at N-terminus as the result of gene cloning were then further purified on a Superdex-75 gel filtration column (Pharmacia). LZ domains were crystallized using the hanging-drop vapor diffusion method.21 Individual hexagonal crystals were grown on siliconized cover slips by equilibrating a mixture containing 1 or 2 μL of protein [5 mg/mL protein in 20 mM Hepes (pH 7.5) and 150 mM NaCl] and 1 μL of reservoir solution against 500 μL of mother liquor [0.1M sodium citrate (pH 5.6) and 20–22% (w/v) PEG8000]. For the cryogenic experiments, 10% glycerol was determined to be a suitable cryoprotectant under the crystallization conditions. Single crystals were soaked in cryoprotection solution for 30 s, mounted on a cryoloop, and flash-frozen in liquid nitrogen. The native data set was collected at beam line BL5A at the Photon Factory. The data set was processed and scaled using the programs DENZO and SCALEPACK.22 The structure of the LZ domain was determined using the molecular replacement technique, with the GCN4 LZ serving as the search model (PDB: 1C94). The program PHASER was used to calculate both the cross-rotation and translation functions in the 10−3 Å resolution range.23 The initial model was subjected to iterative cycles of crystallographic refinement with the program CNS24 that were alternated with graphic sessions for model building using the program O.25 The crystal structure of the LZ domain was refined at 2.1 Å resolution. The asymmetric unit contains two molecules. The trimeric structure of LZ domains is formed by crystallographic threefold symmetry. During the refinement, hemihedral twinning was detected and treated within the program CNS. The twin fraction was 0.35, indicating the crystal to be only partially twinned. A twinning operator was included in the refinement, lowering Rwork and Rfree to 21.0% and 27.8%. The coordinates of the final models and experimental structural factors of the LZ domain have been deposited with the PDB codes 2PNV. The data collection and refinement statistics are summarized in Table I. SKCa LZ domains formed a three-stranded parallel helix bundle shaped like twisted circular cylinder ∼50 Å long and ∼20 Å wide [Fig. 1(A)]. The leucine, isoluecine, and phenylalanine side chains at positions a and d point to the center of the trimer. The first 35 residues of each peptide monomer include more than nine helical turns, though His523 and Ala524 are not in a helical conformation in every chain within the trimer. Leu525 and Pro526 were not visible on the electron density map. (A) Stereo view of the SKCa channel LZ trimer showing heptad residues at a (red) and d (deep blue). (B) Conserved LZ motifs of various channels in vertebrates and a typical LZ motif of a transcriptional factor (SK2, SKCachannel isoform 2; BK, big conductance Ca2+- and voltage-activated K+ channels; CaV1.1, voltage-dependent L-type Ca2+ channel α1S subunit; RyR2, Ryanodine receptor 2; GCN4, yeast transcriptional activator). Residues at position d of the heptad repeats are colored deep blue and residues at position a are colored red. (C) Helical wheel representation of the LZ trimer. Shown are residues 488–526 of SKCa channel isoform 2 (NIMYDMISDLNERSEDFEKRIVTLETKLETLIGSIHALP); the view is from the N-terminus, and residues in the first helical turns are boxed (Ile489) or circled. Heptad positions are labeled a through g. Hydrophobic interactions in the core of the helix bundle at position a and d are connected with blue dashed lines. Hydrogen pairs between Asn498 at position e and Arg500 at position g are indicated by red dashed lines across the interhelical interface. (D) Glutaraldehyde cross-linking assay of LZ domain of the SKCa channel. The purified LZ domain of rat SK2 channel (lane 1: before cross-linking assay) was incubated with 0.1% glutaraldehyde for 30 s (lane 2, 4) or 60 s (lane 3, 5). The concentration of assayed protein was 0.2 mM (lane 2, 3) or 0.6 mM (lane 4, 5). The cross-linking reaction was quenched by adding sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris–HCl pH6.8, 25% glycerol, 2% SDS, 14.4 mM β-mercaptoethanol, 0.1% bromophenol) to the final 1×-working concentration. The cross-linked samples were analyzed by 15% SDS–polyacrylamide gel electrophoresis. The individual helices are smoothly bent, which permits tight contacts over the length of the trimer. The trimer interface shows knobs-into-holes packing between the helices.2, 4 The knobs formed by the side chains of one helix fit into holes formed by the spaces between side chains on the neighboring helix. The isoleucine and leucine knobs at position a pack into a hole formed by the g and a residues of the neighboring monomer. Similarly, residues at positions a and d pack into a hole formed by the d and e residues. In addition, Asn498 and Arg500 contribute to the interaction among the helices through hydrogen bonding and knobs-into-holes packing [Fig. 1(B)]. The trimer interface buries 593 Å2 of protein surface, which represents approximately 17% of the total surface area of each subunit. The residues at positions a, d, e, and g of each strand are largely buried in the α-helix bundle accounting for about 60% of total contact area. The residues at the b and c positions are somewhat buried, and the f position remains completely exposed. Ours is the first description of the three-dimensional crystal structure of an ion channel LZ domain. The overall structure of the α-helix bundle of the SKCa LZ is very similar to the LZ helix bundle in HIV-1 gp41.7 It is interesting that the trimeric parallel α-helix bundle formed by SKCa LZ domains include none of the previously reported residue substitutions.5-7, 26 Some features of the SKCa LZ heptad repeats would appear to facilitate trimer formation. Like the Val residues at position a in GCN4,4 the β-branched Ile494, Ile508 and Ile522 residues at position a should make tetramer formation unfavorable.5, 6 And the Ile508 residue in the SKCa LZ would be more likely to prevent dimer formation than the Asn residue at position a in the GCN4 LZ4 [Fig. 1(C)]. Moreover, hydrogen interactions between Asn498 and Arg500 at positions e and g, respectively, would further stabilize the trimeric structure of the SKCa LZ [dashed line in Fig. 1(C)]. On the other hand, this unique trimeric state of the SKCa LZ domain is inconsistent with the tetrameric nature of the functional SKCa channel. Notably, although X-ray crystallographic analysis confirmed that the SKCa LZ domainforms a three-helix bundle, our chemical cross-linking studies with purified SKCa LZ domain variously suggested that in solution the domain assumes monomeric to tetrameric forms [Fig. 1(D)]. This may be indicative of the important role played by ion channel LZ domains in linking the channels to LZ-containing macromolecular complexes.20 We suggest the apparent flexibility in the binding characteristics of LZ homomers is the major driving force determining specific heteromer formation. Furthermore, the instability of the homomeric states of the SKCa LZ domain in solution suggests that native channel LZs are designed to favor formation of heteromers via LZs of different proteins rather than homomeric structures. The diversity of its interactions highlights the point that within the heptad repeat, the core buried residues at positions a and d or the flanking residues at positions e and g are not sufficient, by themselves, to mediate multimerization. Future crystallization of ion channel LZ domain in complex with a regulatory protein should enable us to visualize the alignment and characteristics of the LZs within the complexes and to further our understanding of their role in modulating ion channel function.
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