Synchrotron Radiation Circular Dichroism Spectroscopy- Defined Structure of the C-Terminal Domain of Nachbac and Its Role in Channel Assembly

Synchrotron radiation circular dichroism spectroscopy- defined structure of the C-terminal domain of NaChBac and its role in channel assembly

Andrew M. Powl, Andrias O. O’Reilly, Andrew J. Miles, and B. A. Wallace1

Department of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom

Edited by Robert Baldwin, Stanford University, Stanford, CA, and approved June 24, 2010 (received for review February 16, 2010)

Extramembranous domains play important roles in the structure channels (7). In the KcsA potassium channel the CTD has been and function of membrane proteins, contributing to protein stabi- shown to be critical for expression, tetramerization, and stability lity, forming association domains, and binding ancillary subunits of the closed channel (8, 9) as well as having a role in pH sensing and ligands. However, these domains are generally flexible, (10, 11). The structure of the CTD of KcsA has recently been making them difficult or unsuitable targets for obtaining high- defined crystallographically (12). It forms a right-handed four- resolution X-ray and NMR structural information. In this study helix coiled-coil bundle of approximately 40 residues in length we show that the highly sensitive method of synchrotron radiation that projects 70 Å beyond the membrane interface, not interact- circular dichroism (SRCD) spectroscopy can be used as a powerful ing significantly with the TM regions of the protein, and is stabi- tool to investigate the structure of the extramembranous C-term- lized by a hydrophobic core in addition to a network of hydrogen inal domain (CTD) of the prokaryotic voltage-gated sodium channel bonds and salt-bridges between adjacent helices. The distal Bacillus halodurans (NaV)from , NaChBac. Sequence analyses pre- section of this CTD adopts a leucine zipper configuration. dict its CTD will consist of an unordered region followed by an Although potassium channels have been well characterized, α-helix, which has a propensity to form a multimeric coiled-coil much less is known about the structural features of voltage-gated motif, and which could form an association domain in the homo- sodium channels (NaVs). However, in 2001 Ren et al. (13) tetrameric NaChBac channel. By creating a number of shortened isolated NaChBac, a simple prokaryotic sodium channel from constructs we have shown experimentally that the CTD does in- Bacillus halodurans that, like potassium channels, but unlike eu- deed contain a stretch of ∼20 α-helical residues preceded by a non- karyotic sodium channels, is composed of a single 6TM domain. helical region adjacent to the final transmembrane segment and Subsequently, many more close homologues from both gram-po- that the efficiency of assembly of channels in the membrane pro- sitive and gram-negative sources have been identified (14–16). In gressively decreases as the CTD residues are removed. Analyses of NaChBac, it has been shown that, as predicted, four monomers the CTDs of 32 putative prokaryotic NaV sequences suggest that a assemble to form an active channel (17). This has prompted the CTD helical bundle is a structural feature conserved throughout the question as to whether a specific assembly domain exists in this bacterial sodium channel family. channel. The ∼40 residues of the CTD of NaChBac at the end of the S6 TM helix represent a candidate region for such a domain, membrane protein assembly ∣ membrane protein structure ∣ synchrotron even though they exhibit negligible sequence identity (∼10% radiation circular dichroism (SRCD) spectroscopy ∣ voltage-gated sodium overall) with the CTD of KcsA. The aim of the present study channel ∣ secondary structure was to determine if the CTD of NaChBac influences expression of this channel and its assembly into the cell membrane fraction odium, calcium, and potassium channels form a family of in- and to elucidate the nature of the CTD. To achieve the latter, we Stegral membrane proteins that selectively regulate ion con- utilized the emerging technique of synchrotron radiation circular ductance across an otherwise impermeable lipid membrane. In dichroism (SRCD) spectroscopy (18), which provides the sensi- eukaryotic voltage-gated sodium and calcium channels the func- tivity and accuracy needed to examine the structures of a series tional unit is formed by assembly of four homologous domains of of closely related truncated constructs of the channel. As a result, a single polypeptide chain. Each domain is composed of six trans- this study has also exemplified a new range of possible applica- membrane (TM) helices, with the four N-terminal helices form- tions of SRCD spectroscopy in structural biology. ing the voltage-sensing subdomain and the two C-terminal helices comprising the pore subdomain. In contrast, in both eukaryotic Results and prokaryotic potassium channels the functional unit is a Expression and Assembly. The prokaryotic sodium channel homo-tetramer formed from identical monomers that each cor- NaChBac shows strong sequence homology to 6TM bacterial and respond to one of the sodium or calcium channel domains (1). eukaryotic potassium channels; in addition to the TM voltage The crystal structures of a number of potassium channels have sensor and pore regions, it also contains a CTD, consisting of been determined (2–4), which have confirmed the tetrameric nature of the channels and details of their transmembrane (TM) Author contributions: A.M.P., A.O.O., and B.A.W. designed research; A.M.P., A.O.O., A.J.M., regions. The structures of the extramembranous C-terminal and B.A.W. performed research; A.M.P., A.O.O., A.J.M., and B.A.W. analyzed data; A.M.P., domains of most of these channels, however, were not defined A.O.O., A.J.M., and B.A.W. wrote the paper; and B.A.W. supervised the project. in the crystal structures, either because they had been removed The authors declare no conflict of interest. after assembly (KcsA) (2) in order to improve crystallization, This article is a PNAS Direct Submission. or because they were disordered in the crystal (KvAP and Freely available online through the PNAS open access option. MlotiK1) (3, 4). Data deposition: The SRCD spectra have been deposited in the Protein Circular Dichroism The extramembranous termini of potassium channels appear Data Bank, http://pcddb.cryst.bbk.ac.uk/ (PCDDB reference nos. NB001AA00, NB001AB00, to play important roles in channel assembly, primarily as tetra- and NB001AC00 for wild-type, delta 239, and delta 254 proteins, respectively). merization domains. Examples include the N-terminal T1 domain 1To whom correspondence should be addressed. E-mail: [email protected]. Shaker of channels (5) and C-terminal domains (CTDs) of the This article contains supporting information online at www.pnas.org/lookup/suppl/ ether-a-go-go (EAG) (6) and the inwardly rectifying (KIR) doi:10.1073/pnas.1001793107/-/DCSupplemental.

14064–14069 ∣ PNAS ∣ August 10, 2010 ∣ vol. 107 ∣ no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1001793107 Downloaded by guest on October 5, 2021 ∼40 residues that extend beyond the TM domain. In order to unlikely to be a result of altered accessibility of the His-tag used identify the role (if any) these residues play in channel assembly for binding of the antibody because the tag is located at the N and structure, a series of truncated channel constructs was pro- terminus, distal to the C terminus; indeed removal of C terminus duced, in which residues were removed consecutively from the C might even be expected to increase accessibility and hence stain- terminus (Fig. 1A). Western blots (Fig. 2 A and B) of the total cell ing. The reduced levels of assembled proteins detected in the lysates and the membrane fractions, respectively, were used to membrane fraction are therefore likely to arise because removal monitor the total amount of NaChBac produced and the amount of the CTD leads to a protein that is either unstable or one that is of protein that assembles into the membrane as the CTD is misfolded and located in the inclusion bodies. The latter is the truncated. most reasonable explanation because these proteins would be de- Firstly, as a positive control, total protein expression was tectable in the whole cell lysate but not in the membrane fraction. shown to be virtually abolished when a stop codon was introduced This is similar to the pattern of expression observed for the KcsA at position 231 (construct designation 231Δ), a mutation de- potassium channel in which removal of the C-terminal-most 36 signed to disrupt the S6 helix since the final ca. four residues residues led to a limited reduction in expression of the channel, of this helix are also removed in addition to ones in the CTD. while removal of the next five residues from its extramembranous Next, residues between 274 (full-length) and 254 (i.e., constructs domain significantly reduced expression (8, 9). 262Δ and 254Δ) were removed; in these cases the total amount of These expression studies thus indicate that the CTD is impor- protein produced was essentially indistinguishable from the wild tant for expression of properly targeted and assembled NaChBac. type. Even when the residues from 247 to 238 were progressively removed, there was only a very modest reduction in the total level Structure. Sequence-based secondary structure predictions of the of expression (Fig. 2B). When the membrane fractions were ex- S6 helix and the CTD of NaChBac using five algorithms (Fig. 1A) amined, the amounts of protein for constructs 262Δ, 254Δ, 247Δ, were used to produce a consensus secondary structure for and 243Δ were found to be comparable to those in the total NaChBac, and the DISOPRED algorithm was used to indicate lysate; however, there was a progressive reduction in the amount regions with high propensities for forming natively disordered of protein detected as residues from 242 to 235 were deleted. structures (Fig. 1A). Together they indicate that both the putative Reductions in the total levels of protein may be a result of S6 TM region (as expected) and the last ca. 22 residues of the C altered RNA stability, decreased protein synthesis, or altered terminus have a high propensity for forming helical structures, protein stability. Because the 240Δ, 239Δ and 238Δ constructs while the connecting 14 residues between S6 and the terminal appeared to be produced in roughly similar amounts as the 22 residues are predicted to be nonhelical (Fig. 1B). wild-type channel when the total cell lysate was assayed, this sug- Synchrotron radiation circular dichroism spectroscopy (Fig. 3) gests that neither altered RNA stability nor decreased protein was used to experimentally determine the secondary structures of synthesis are key factors in the reduction of the amount of protein the various constructs and compare them with the predicted in the membrane-associated fractions. In addition, the reduction helical contents for these constructs (Table 1). The use of SRCD, in intensity of the Western blot bands of the shorter constructs is a newly emerging tool for biophysical characterization, has been BIOCHEMISTRY SCIENCES APPLIED PHYSICAL

Fig. 1. (A) Predicted secondary structure of NaChBac CTD based on its primary sequence using five algorithms. Code: (H) helix, (E) sheet, (N) nonhelical (random coil), or (-) not assigned. Predicted disordered regions are indicated by *. The coiled-coil prediction was done using sliding windows of either 14 or 21 residues, designated Lupas-14 and Lupas-21, respectively. The sites of truncation of the various constructs are indicated by vertical black arrows above the sequence, and the sites of the point mutations are indicated below the sequence by °. At the bottom of the figure is a hydrophobicity plot of the same region (residues 206 to 274), generated using the DNASTAR suite of programs. A window of 19 (solid line) was used for identifying TM segments, and a window of 9 (filled in shaded regions) was used for identifying leucine zipper features. (B) Homology model for the NaChBac pore (blue) and CTD (cyan) regions, starting at the N terminus of the S5 helix, with arrows showing the approximate positions of the constructs examined by SRCD spectroscopy. The green arrows indicate the incrementally removed residues were helical, and the red arrows indicate they were nonhelical.

Powl et al. PNAS ∣ August 10, 2010 ∣ vol. 107 ∣ no. 32 ∣ 14065 Downloaded by guest on October 5, 2021 Calculations indicate (Table 1) that when the 36 amino acids between positions 274 and 239 were removed, the total number of α-helical residues lost was ∼22, a strong correspondence with the predicted value; furthermore essentially all of the α-helical residues lost specifically involved the C-terminal-most residues between 274 and 254 (Table 1). The SRCD studies of constructs missing the additional residues between 247 and 239 showed essentially no further loss of helical residues, also in accord with the sequence-based secondary structure predictions. This then suggests the distal C-terminal residues are helical, but that the residues closest to the TM segments are nonhelical (Fig. 1B). Hydropathy plots of the CTD of NaChBac indicate the sequence corresponding to the ∼22 amino acid C-terminal helical region has a typical repeat pattern of hydrophobic and hydrophilic residues found in coiled-coil structures (Fig. 1A). Coiled- Fig. 2. Western blot analyses of the expression of the wild-type NaChBac E. coli coil proteins have a characteristic seven residue repeat of and CTD truncation constructs. The same number of cells (as deter- ð Þ mined by OD600) were used in each case for (A) whole cell extracts or (B) a:b:c:d:e:f:g n, with helix interactions driven by hydrophobic in- isolated membranes. Constructs are numbered according to the position teractions of the residues at the a and d positions (22). In soluble of the stop codon along the NaChBac sequence. For example, in construct coiled-coil proteins leucines occupy ca. 80% of all d positions and 231Δ the C-terminal residue is 230. these structures are often referred to as leucine zippers (23). For the stretch of α-helical residues located at the C terminus of NaChBac, all three d positions are occupied by leucine residues, essential for these studies due to its high signal-to-noise levels and whereas the a positions are occupied by either leucine, isoleucine, hence high sensitivity to very small structural changes (19, 20). All or valine. Residues 249–274 plotted as a helical wheel (Fig. S1A) of the SRCD spectra of the constructs are very similar in shape, show a single face of the wheel is hydrophobic, with smaller or indicative of proteins with high helix contents, but differences in charged/polar amino acids on the remaining surfaces. When four their spectra are reflective of their resulting from proteins with helical wheels are placed into a parallel tetrameric arrangement subtly different helical contents. The experimental error bars (Fig. S1B), the resultant structure has a central hydrophobic core (one standard deviation) seen in the inset to Fig. 3 show that (Fig. S1C) and could possess intersubunit salt bridges formed the high precision of the measurements makes it possible to dis- between neighboring helices, e.g., K253-E255 and R260-D262, tinguish with significance the spectra of these very closely related which could also contribute to the overall stability of the structures; furthermore, their reproducibility was demonstrated coiled-coil structure. The observation (12) of a similar type of by comparisons of measurements on different samples of the structural element in the CTD of the full-length KcsA channel same construct at two different beamlines (Soleil in France (albeit without the linking nonhelical region attaching it to S6) and ISA in Denmark), where the differences are ≤ the error bars sets a precedent for such an ion channel tetramerization domain for repeated scans of a single preparation (data not shown). The composed of a coiled-coil four-helix bundle. Together these predictions and experimental observations sug- increased accuracy in secondary structure determinations using gest the CTD of NaChBac begins at the end of TM6 with a non- SRCD, when the low wavelength vacuum ultraviolet data (not helical region that links the TM pore with a C-terminal coiled-coil measurable with conventional CD instruments) is included [it helical bundle. has been shown to produce correlation coefficients for helical components between crystal and SRCD-derived structures of Tetramerization. Previous studies on the removal of the CTD from 0.956 (21)], means that in this study it has been possible to iden- KcsA indicated that the protein migrates as a tetramer (as does tify secondary structural differences on the order of 1% (Table 1) the full-length KcsA) on SDS gels, suggesting that once the tetra- associated with the very small differences in protein constructs. mer is assembled the channel is extremely stable in vitro, to both detergent and heat-induced dissociation (8, 9, 11). Unlike KcsA, the full-length NaChBac tetramer is not resistant to dissociation by SDS but instead runs as a monomer on SDS denaturing gels (Fig. 2); therefore, in order to determine the yield of oligomers in the membrane fraction, we monitored the gel-filtration (GF) profile of the isolated channels solubilized in the nondissociating detergent Cymal-5 (Fig. 4). The GF data indicated that wild-type channel (plus the His- tag) had a peak elution volume corresponding to a calculated size of ∼210 kDa (protein plus detergent), as we had found earlier (17). This indicates that most of the wild-type channel is tetrameric, with no monomeric protein detected; a small population was observed to exist as aggregates, eluting at the void volume (Fig. 4). The shortest construct, 239Δ, eluted at a peak volume corresponding to a calculated molecular weight of ∼190 kDa. A shift in the peak elution volume for this construct is in agreement with it being smaller in size than the wild-type channel and Fig. 3. Synchrotron radiation circular dichroism (SRCD) spectra of NaChBac consistent with the fact that 36 residues in each of its monomers protein constructs in the detergent Cymal-5. Comparison of the SRCD spectra (total ∼16 kDa) had been removed. However, the total yield of of NaChBac (solid line) and CTD-truncated channels 254Δ (dotted line) and Δ Δ Δ tetrameric channel isolated for this construct (despite starting 239 (dashed line). The spectra of the 262 and 247 constructs were inter- 80% mediate and not shown for clarity. Inset: expansion of the wavelength region with the same amount of membranes) was reduced by > with between 185 nm and 200 nm showing error bars (at one standard deviation respect to wild type, consistent with the relative amounts seen on in the measurement). the Western blots. Constructs 247Δ, 254Δ and 262Δ also eluted in

14066 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001793107 Powl et al. Downloaded by guest on October 5, 2021 Table 1. Predicted and experimentally determined secondary structures of the different CTD constructs of NaChBac No. residues No. α-helical residues Helical secondary structure No.α-helical residues calculated Construct removed predicted missing determined from SRCD spectra (%) missing from SRCD spectra wild-type (274) 0 0 66.4 0 262Δ 13 11 64.8 12 254Δ 21 20 63.3 22 247Δ 28 21 64.8 22 239Δ 36 21 66.6 23

comparably diminished yields but with retention volumes inter- The nonhelical region linking S6 and the putative helical bundle mediate between that of the wild-type channel and the 239Δ con- appears to be particularly important for the assembly process. It struct (not shown for clarity). contains a large number of negatively charged residues and a Hence, it appears that while the yield of membrane-associated single positively charged residue (K237) (Fig. 1A). These polar channels decreased with removal of the CTD, all channels iso- residues, located in proximity to the TM-cytoplasmic interface, lated from the membrane fraction are tetrameric and remain may form interactions with residues on the S5 helix or S4-S5 lin- so even upon solubilization. This suggests that the CTD influ- ker that are necessary for the correct folding of the channel. Ala- ences the assembly but not necessarily the stability of the quatern- nine scanning mutagenesis studies (Fig. S3) has revealed that a ary structure. number of the residues in this region, especially K237, are highly sensitive to mutation, with single changes greatly reducing or Homologues. BLAST searches identified 32 putative prokaryotic eliminating assembly of full-length channels in the membrane. NaV sequences, a number of which had not previously been iden- Such a highly charged region may also be a common feature tified as members of this family. Sequence alignments of their in the other prokaryotic sodium channels, because most of the CTDs (Fig. S2) suggest that all of these homologues possess a homologue sequences display a high proportion of negative resi- stretch of residues equivalent to at least four turns of a helix com- dues and a single positive residue at roughly the same positions prising at least two heptad repeats. This high level of conservation with respect to the water-lipid interface in this region of the CTD of the motif indicates that an intersubunit coiled-coil helical (Fig. S2). We speculate that the role of the lysine residue, which bundle may be an important structural feature in the bacterial when mutated essentially abolishes assembly in the membrane, sodium channel family. may either be to form specific interactions with the head groups of the lipid molecules (26) or an intersubunit salt bridge or may Discussion simply be required to satisfy the positive-inside rule (27) for mem- In the C-terminal truncated constructs, the levels of channel pro- brane protein insertion. tein produced by the cell (as determined from whole cell lysate) The presence of association domains may be common structur- did not appear to be significantly altered, while the amount of al features for tetrameric members of the family of voltage-gated channel protein located in the membrane fraction was apparently ion channels. Our sequence analyses suggest that similar structur- reduced as the length of the CTD decreased. Once the CTD was al features may be present in all (or most) prokaryotic sodium fully deleted, no channels could be detected in the membrane channels. Previous studies with KIR and EAG potassium channels fraction. demonstrated the necessity of a tetramerization domain for chan- SRCD spectroscopy has shown that approximately 22 amino nel expression (5–7, 9). In some cases the domain is located at the acids at the C-terminal end are helical in nature, but that there N terminus (5), and in some cases at the C terminus, of the TM is also a novel nonhelical section between the end of the S6 helix regions (6, 7). In KcsA, the CTD is also ∼40 residues in length but and the helical C-terminal residues. Sequence predictions suggest is virtually entirely helical in the closed-state structure (12) and the 22 helical residues may form an intersubunit coiled-coil motif. shares only ∼10% sequence identity with the NaChBac. Hence it The CTD may contribute to the assembly of the tetramer. How- appears that it is the presence of a domain that can aid associa- BIOCHEMISTRY ever, once a tetramer is formed, other stabilizing interactions tion, rather than the details of the domain structure, that is the such as van der Waals, hydrogen-bonding, electrostatic, and common feature among channels in this family. Tetramerization aromatic-aromatic interactions elsewhere in the molecule may domains presumably aid channel assembly by recruiting nascent also contribute to the stability of the tetrameric channel (24, 25). monomeric chains to an oligomeric ensemble, allowing physical convergence of the TM regions. Structural rearrangements of the TM domains may then be required to form the functional product once these regions are brought into close proximity. SCIENCES

In summary, we have used the highly sensitive method of APPLIED PHYSICAL SRCD in conjunction with bioinformatics predictions to propose a structure for the CTD of NaChBac at the residue level, consisting of a four-helix coiled-coil bundle with a leucine zipper configuration preceded by a highly charged nonhelical region adjacent to the S6 helix. Further, we suggest that the CTD plays an important role in channel expression and assembly and may be a common familial feature in all prokaryotic sodium channels. This study has also demonstrated the utility of SRCD spectroscopy for characterizing proteins. In order to produce the level of detail described, high levels of precision, reproducibility, and accuracy not ordinarily achievable with conventional CD spectroscopy, but possible with SRCD, were required. The sodium chan- Fig. 4. Gel-filtration characterization of wild-type NaChBac (solid line) and the 239Δ construct (dashed line) in the detergent Cymal-5. Both constructs nel has been ideal for exemplification of the new application run with molecular masses corresponding to tetramers, with the 239Δ because its CTD domain is dominated by helical features, which mutant running slightly slower than the wild type due to the deletion of are very accurately determined by SRCD spectroscopy, and 36 residues in each of its monomers. because its CTD is essentially a separate domain, so removals

Powl et al. PNAS ∣ August 10, 2010 ∣ vol. 107 ∣ no. 32 ∣ 14067 Downloaded by guest on October 5, 2021 of individual amino acids do not alter the bulk of the protein re- protein, the extinction coefficient at 280 nm was calculated from the amino maining. This type of application of SRCD adds a new dimension acid sequence using the Expasy ProtParam website (29) and the concentra- to our ability to characterize protein structural domains. tion determined from the A280 measured on a Nanodrop 1000 UV spectro- photometer in triplicate immediately before the SRCD spectrum was Materials and Methods acquired. The values thus determined are very reproducible (variation of less than þ∕−1%) and have been shown to be highly correlated with quantita- Materials. The cDNA for NaChBac was generously provided by David Clapham of Harvard Medical School. N-dodecyl-β-D maltopyranoside and 5-cyclohexyl- tive amino acid determinations of protein concentration (30). Three SRCD ∼1 ∕ 1-pentyl-β-D maltoside (Cymal-5) were obtained from Anatrace. All other spectra were collected for each protein sample [ mg ml in a 0.054 mm reagents were purchased from Sigma, unless otherwise stated. pathlength Suprasil demountable cell (Hellma)] at 4 °C over the wavelength C-terminal truncated NaChBac constructs were generated using the range from 260 nm to 175 nm, with a 1 nm step size and a 1 s dwell time. QuikChange protocol from Stratagene to introduce stop codons into the Three baseline spectra (which consisted of the GF column flow-through) were full-length NaChBac cDNA (corresponding to 274 amino acids) that had pre- averaged and subtracted from the averaged sample spectrum, and the varia- viously been cloned into expression vector pET15b (17). Constructs are num- bility (one standard deviation) in the experimental measurements calculated bered according to the position of the stop codon along the NaChBac at each wavelength. The spectra were smoothed with a Savitsky-Golay filter, sequence. For example, construct 231Δ has the sequence of DNA encoding and scaled to delta epsilon values using mean residue weights of 114.4, 114.5, Δ Δ Δ the amino acid at 231 replaced by a stop codon so that the C-terminal residue 114.4, 115.0, 114.9, respectively, for the wild-type, 262 , 254 , 247 , Δ is now 230. All mutations were confirmed by sequencing on both strands. and 239 constructs. Data processing was carried out using the CDTools software (31). Data were analyzed with the DichroWeb analysis server (32). The reported values are the average results from the CONTINLL, SELCON3, and Membrane Preparations and Western Blots. Western blots were performed on either whole cell lysate or on isolated membrane preparations from cells. CDDSTR (33) algorithms calculated using the SP175 reference dataset (21). Single colonies of Escherichia coli C41(DE3) transformants harboring the pET15b plasmid with the NaChBac genes were grown in Luria broth supple- Sequence Analysis and Molecular Modeling. The NaChBac sequence was used mented with ampicillin (100 μg∕ml) at 37 °C. Upon reaching an OD600 of ca. in a BLASTP search of the nonredundant protein database (34) to identify 32 0.7, cultures were induced with 0.3 mM isopropyl-β-D thiogalactopyranoside putative prokaryotic NaVs based upon inclusion of the characteristic posi- at 37 °C for 1 h. An aliquot of cells equivalent to an OD600 of 2.0 was then tively charged residues in the S4 voltage-sensing helix followed by a TxExW pelletted at 6;000 × g for 5 min. For samples of whole cell lysate the pellet sequence in the filter region (in NaChBac this is TLESW) (13, 14). was resuspended in NuPAGE lithium dodecyl sulfate (LDS) sample buffer. The secondary structure of the NaChBac CTD from the N-terminal end of Alternatively, to isolate cell membranes, the cell pellet was resuspended in the S6 helix onward was predicted using PSIPRED (35), JPred3 using PSIBLAST 150 mM NaCl, 20 mM Tris·HCl, pH 7.8 (TBS) and lysed by sonication. Lysed and HMMER2 profiles (36), the HNN Hierarchical Neural Network method cells were spun at 6;000 × g for 5 min and the supernatant spun at 100;000 × (37); PHD, (38) and APSSP2 (39). The DISOPRED algorithm (part of PSIPRED) g for a further 20 min. Membrane pellets were resuspended and solubilized was used to indicate regions with high propensities for forming natively dis- by aspiration in TBS supplemented with NuPAGE LDS sample buffer. ordered structures. The program Coils (version 2.2) (40) was used for the Proteins from whole cell lysates or membrane fractions were separated on prediction of coiled-coil domains. Sequence alignments of the 32 putative a precast denaturing gel (NuPAGE 4–12% gradient Bis-Tris gel w/MOPS) and prokaryotic NaVs were carried out using the ClustalW algorithm (41) and transferred to a polyvinyl difluoride membrane. Monoclonal anti-polyHis- the sequence corresponding to the CTD immediately carboxyl to the S6 pore alkaline phosphatase antibody was used to identify the His-tagged proteins helix was manually aligned for maximal hydrophobicity at positions a and d and immunoblotting signals were developed by BCIP/NBT FAST tablets. of the heptad sequence. An homology model of NaChBac was generated as described in O’Reilly et Protein Purifications and Gel-Filtration Measurements. Protein samples were al. (28), except using the full-length KcsA crystal structure (12) as template – – purified from E. coli C41(DE3) cells expressing the NaChBac genes, as des- (PDB code 3EFF). Residues 207 237 and 251 274 of the NaChBac sequence – – cribed previously (28), with the following modification; protein was eluted were aligned, respectively, with residues 87 117 and 136 159 of KcsA to gen- in 0.3% Cymal-5, 150 mM NaCl, 20 mM sodium phosphate, pH 7.8 containing erate the S6 and CTD helical sections. The 14-residue nonhelical section of the 300 mM imidazole and concentrated in an Amicon concentrator (either a NaChBac CTD was modeled using the loop function of the SYBYL Biopolymer 100 KDa or 10 KDa cut-off). The sample was spun at 100;000 × g at 4 °C module (Version 8.0, Tripos Inc., St. Louis). Fig. 1B was produced using the for 20 min and loaded onto a size-exclusion GF column (Superdex 200 PyMOL molecular graphics system (DeLano Scientific, San Carlos, CA). 10∕300; GE Healthcare) equilibrated with 50 mM NaCl, 20 mM sodium phosphate, pH 7.8 containing 0.3% Cymal-5 at 4 °C and eluted at a rate of ACKNOWLEDGMENTS. We thank Dr. Robert W. Janes (Queen Mary, University 0.3 ml∕ min. The GF column had been calibrated with thyroglobulin, ferritin, of London) for help with SRCD data collection. We would like to thank amylase, adolase, alcohol dehydrogenase, covalbumin, ovalbumin, carbonic Dr. Frank Wien for assistance at the Soleil Synchrotron DISCO beamline anhydrase, and cytochrome. The fractions corresponding to the expected and Dr. Soren Vronning Hoffmann for assistance at the ISA Synchrotron tetrameric channel molecular mass were then run on a precast denaturing CD1 beamline. This work was supported by grants from the U.K. Biotechnol- gel (NuPAGE 4–12% gradient Bis-Tris gel w/MOPS). ogy and Biological Research Council and the Heptagon Fund and by the VIP programme of the Wellcome Trust. SRCD access to both the Soleil and ISA Synchrotrons was supported by beamtime grants to B.A.W. Access at ISA Synchrotron Radiation Circular Dichroism Spectroscopy. SRCD spectra were is acknowledged under the EU Integrated Infrastructure Initiative, Integrated collected on the DISCO beamline at the Soleil Synchrotron, France, and Activity on Synchrotron, and Free Electron Laser Science (IA-SFS), contract repeated on the CD1 beamline at the ISA Synchrotron, Denmark. For each number RII3-CT-2004-506008.

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