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

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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 nat- ure 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.
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