Published on Web 08/04/2005
Bead-linked Proteoliposomes: A Reconstitution Method for NMR Analyses of Membrane Protein-Ligand Interactions Mariko Yokogawa,† Koh Takeuchi,† and Ichio Shimada*,†,‡ Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Biological Information Research Center (BIRC), National Institute of AdVanced Industrial Science and Technology (AIST), Aomi koto-ku, Tokyo 135-0064, Japan Received February 24, 2005; E-mail:
[email protected] Abstract: Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands.
Introduction
Protein-protein interactions are essential in many biological processes, such as signal transduction, cellular recognition, and the immune response. In particular, membrane proteins, which represent 20-30% of the total proteins encoded by the human genome,1 play important roles through their interactions with intra- and extracellular ligands and the transmission of information across membranes. Even with the recent expansion of structural genomics2-5 and the general appreciation of their biological significance, little is known about the structures and functions of membrane proteins. This is mainly due to practical problems in handling membrane proteins. One common problem is the poor stability of membrane proteins, under conventional experimental conditions. Although surfactant-solubilized membrane proteins have been used in structural analyses,6 the solubilization of membrane proteins frequently causes conformational instability, followed by aggregation or denaturation.7,8 Furthermore, the instability renders many membrane proteins biochemically intractable and precludes high-resolution structure determinations. †
The University of Tokyo. National Institute of Advanced Industrial Science and Technology (AIST). ‡
(1) Wallin, E.; von Heijne, G. Protein Sci. 1998, 7, 1029-1038. (2) Yokoyama, S.; Hirota, H.; Kigawa, T.; Yabuki, T.; Shirouzu, M.; Terada, T.; Ito, Y.; Matsuo, Y.; Kuroda, Y.; Nishimura, Y.; Kyogoku, Y.; Miki, K.; Masui, R.; Kuramitsu, S. Nat. Struct. Biol. 2000, 7 Suppl, 943-945. (3) Terwilliger, T. C. Nat. Struct. Biol. 2000, 7 Suppl, 935-939. (4) Heinemann, U. Nat. Struct. Biol. 2000, 7 Suppl, 940-942. (5) Burley, S. K. Nat. Struct. Biol. 2000, 7 Suppl, 932-934. (6) Sanders, C. R.; Oxenoid, K. Biochim. Biophys. Acta 2000, 1508, 129145. (7) Bowie, J. U. Curr. Opin. Struct. Biol. 2001, 11, 397-402. (8) Zhou, Y.; Lau, F. W.; Nauli, S.; Yang, D.; Bowie, J. U. Protein Sci. 2001, 10, 378-383. 10.1021/ja0511772 CCC: $30.25 © 2005 American Chemical Society
It is generally accepted that membrane proteins maintain their native conformation more stably in lipid bilayers than in a surfactant-solubilized environment,9-11 and some membrane proteins are known to require specific phospholipids to maintain their structure and function.12,13 Therefore, in molecular biological analyses, biomembranes where membrane proteins of interest are inherently expressed or engineered to be expressed have been used. In contrast, several membrane-protein-reconstitution techniques for investigating the functions of membrane proteins, such as liposome incorporation14 and reconstitution on chips15,16 and on beads,17 have been utilized to artificially reconstitute membrane proteins into lipid bilayers. However, primarily due to the heterogeneity of the reconstituted samples, containing lipid bilayers and membrane proteins, it is difficult to apply these strategies to structural analyses using X-ray crystallography. Although NMR, another powerful tool for structural analyses of biomolecules, tolerates targeting-system heterogeneity, it has not been recognized as an ideal tool for structural analyses of membrane proteins. The main drawback is its low (9) van Dalen, A.; Hegger, S.; Killian, J. A.; de Kruijff, B. FEBS Lett. 2002, 525, 33-38. (10) van den Brink-van der Laan, E.; Chupin, V.; Killian, J. A.; de Kruijff, B. Biochemistry 2004, 43, 4240-4250. (11) Engel, C. K.; Chen, L.; Prive, G. G. Biochim. Biophys. Acta 2002, 1564, 47-56. (12) Lange, C.; Nett, J. H.; Trumpower, B. L.; Hunte, C. Embo J. 2001, 20, 6591-6600. (13) Jordan, P.; Fromme, P.; Witt, H. T.; Klukas, O.; Saenger, W.; Krauss, N. Nature 2001, 411, 909-917. (14) Devesa, F.; Chams, V.; Dinadayala, P.; Stella, A.; Ragas, A.; Auboiroux, H.; Stegmann, T.; Poquet, Y. Eur. J. Biochem. 2002, 269, 5163-5174. (15) Stenlund, P.; Babcock, G. J.; Sodroski, J.; Myszka, D. G. Anal. Biochem. 2003, 316, 243-250. (16) Karlsson, O. P.; Lofas, S. Anal. Biochem. 2002, 300, 132-138. (17) Mirzabekov, T.; Kontos, H.; Farzan, M.; Marasco, W.; Sodroski, J. Nat. Biotechnol. 2000, 18, 649-654. J. AM. CHEM. SOC. 2005, 127, 12021-12027
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sensitivity, which necessitates the use of large amounts of purified membrane proteins, and the molecular-weight limitations in NMR measurements. In a conventional NMR analysis, the molecular weight of the target protein has to be less than 150 kDa18,19 and the protein concentration should ideally be more than 1 mM. Although several surfactant-solubilized membrane proteins have been successfully analyzed,20-23 membrane proteins reconstituted in lipid bilayers obviously exceed the molecular weight limitations. Furthermore, the low concentrations achieved by conventional reconstitution strategies are insufficient for carrying out an extensive structural analysis. We recently proposed a novel NMR method, termed transferred cross-saturation (TCS), for identifying contact residues in a large protein complex.24 In this method, the molecular weight limitation is overcome by properly transferring information about the contact residues on the ligand proteins in the complex, from the ligand proteins in the bound state to those in the free state, under a fast exchange process. We have already demonstrated the effectiveness of this method for investigating the protein interactions of extremely large proteins, liposomes, and insoluble biomolecules.25-27 However, the development of a sample preparation strategy that ensures the incorporation of a sufficient amount of membrane protein into lipid bilayers has remained elusive. Here, we propose a novel strategy that reconstitutes membrane proteins linked to beads, where the membrane proteins are embedded in lipid bilayers. In combination with the TCS method, the bead-linked proteoliposomes facilitate the identification of the contact residues on ligand proteins interacting with a wide variety of membrane proteins. In the present paper, we prepared bead-linked proteoliposomes, composed of a potassium channel, KcsA, embedded in the lipid bilayer (KcsA-proteoliposomes), and used them to investigate the interaction between KcsA and its pore-blocking peptide, agitoxin2 (AgTx2). Analyses of the KcsA-proteoliposomes with the TCS method allowed us to successfully identify the KcsA binding surface on AgTx2. Materials and Methods Materials. Ni-NTA silica beads were purchased from QIAGEN. All phospholipids, including the fluorescently labeled phospholipids, were obtained from Avanti Polar Lipids as chloroform solutions. Oregon green 488 maleimide (OGM) was purchased from Molecular Probes Inc. Other chemicals were obtained from Wako Chemicals, Nacalai (18) Salzmann, M.; Pervushin, K.; Wider, G.; Senn, H.; Wuthrich, K. J. Am. Chem. Soc. 2000, 122, 7543-7548. (19) Tugarinov, V.; Muhandiram, R.; Ayed, A.; Kay, L. E. J. Am. Chem. Soc. 2002, 124, 10025-10035. (20) Arora, A.; Abildgaard, F.; Bushweller, J. H.; Tamm, L. K. Nat. Struct. Biol. 2001, 8, 334-338. (21) Oxenoid, K.; Kim, H. J.; Jacob, J.; Sonnichsen, F. D.; Sanders, C. R. J. Am. Chem. Soc. 2004, 126, 5048-5049. (22) Fernandez, C.; Hilty, C.; Bonjour, S.; Adeishvili, K.; Pervushin, K.; Wuthrich, K. FEBS Lett. 2001, 504, 173-178. (23) Hwang, P. M.; Choy, W. Y.; Lo, E. I.; Chen, L.; Forman-Kay, J. D.; Raetz, C. R.; Prive, G. G.; Bishop, R. E.; Kay, L. E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13560-13565. (24) Nakanishi, T.; Miyazawa, M.; Sakakura, M.; Terasawa, H.; Takahashi, H.; Shimada, I. J. Mol. Biol. 2002, 318, 245-249. (25) Takeuchi, K.; Takahashi, H.; Sugai, M.; Iwai, H.; Kohno, T.; Sekimizu, K.; Natori, S.; Shimada, I. J. Biol. Chem. 2004, 279, 4981-4987. (26) Takeuchi, K.; Yokogawa, M.; Matsuda, T.; Sugai, M.; Kawano, S.; Kohno, T.; Nakamura, H.; Takahashi, H.; Shimada, I. Structure (London) 2003, 11, 1381-1392. (27) Nishida, N.; Sumikawa, H.; Sakakura, M.; Shimba, N.; Takahashi, H.; Terasawa, H.; Suzuki, E. I.; Shimada, I. Nat. Struct. Biol. 2003, 10, 5358. 12022 J. AM. CHEM. SOC.
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Tesque, and Dojindo. Expression, purification, and stable-isotope labeling of KcsA and AgTx2 were carried out as described.26 Preparation of Zn-NTA Beads. NTA silica beads were utilized as the base of the KcsA-proteoliposomes. After removal of the prechelated Ni2+ ions from the Ni-NTA silica beads with 0.3 mM ethylenediaminetetraacetic acid (EDTA) and extensive washing with H2O, the beads were charged with Zn2+ ions by an incubation in 100 mM ZnSO4. The excess Zn2+ ions were removed with H2O to create the Zn-NTA beads. Preparation of Lipid Solutions. 1-Palmitoyl-2-oleoyl-sn-glycero3-phosphoethanolamine (POPE) or 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), mixed with 0.2% (by weight) 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (rhodamine-DOPE), was dried by rotary evaporation using a roundbottomed flask to yield a thin lipid film. The film was then thoroughly dried under a vacuum for at least 3 h. Buffer A, containing 20 mM Tris-HCl (pH 8.0) and 150 mM KCl, was added to the lipid film and vortexed vigorously under a nitrogen atmosphere, which typically yielded a 16 mg/mL lipid solution. The lipid solution was solubilized by adding 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), at final concentrations of 120 mM for POPE and 85 mM for POPC. Construction of the KcsA-Proteoliposomes. The purified KcsA solution (20 µM) was incubated with the Zn-NTA beads in a 1.5 mL tube for 2 min. After mild centrifugation, the supernatant was removed. These processes were repeated until the desired amount of KcsA was immobilized on the beads. After the immobilization procedure, n-dodecyl-β-D-maltoside (DDM) was exchanged for CHAPS. This exchange procedure might be important for the successful organization of the phospholipid bilayers on the beads, because the higher critical micelle concentration (CMC) of CHAPS (6-10 mM) would be more advantageous for efficient dialysis than the lower CMC of DDM (
