Structural Properties of AMP-activated Protein Kinase
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m708379200
ISSN1083-351X
AutoresUwe Riek, Roland W. Scholz, Petr V. Konarev, Arne C. Rufer, Marianne Suter, Alexis Nazabal, Philippe Ringler, Mohamed Chami, Shirley A. Müller, Dietbert Neumann, Michael Forstner, Michael Hennig, Renato Zenobi, Andreas Engel, Dmitri I. Svergun, Uwe Schlattner, Theo Wallimann,
Tópico(s)Plant nutrient uptake and metabolism
ResumoHeterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing γ1, (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (∼1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by ∼5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr172, thus positively affecting AMPK activity. Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing γ1, (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (∼1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by ∼5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr172, thus positively affecting AMPK activity. Mammalian AMP-activated protein kinase (AMPK) 4The abbreviations used are: AMPK, AMP-activated protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; CBS, cystathionine β synthase; HPLC, high pressure liquid chromatography; IMAC, immobilized metal ion affinity chromatography; ITC, isothermal titration calorimetry; LKB1, serine/threonine kinase 11 (STK11); MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MO25, mouse protein 25; MS, mass spectrometry; RS, regulatory sequence; SAMS, synthetic peptide HMR-SAMSGLHLVKRR; SAXS, small angle x-ray scattering; SNF1, carbon catabolite-derepressing protein kinase; STEM, scanning transmission electron microscopy; STRAD, STE20-related adaptor protein; TEM, transmission electron microscopy; Ni-IDA, nickel iminodiacetic acid resin. and its orthologs found in yeast, plants, insects, invertebrates, and vertebrates are fuel sensors of the eukaryotic cell and function as master regulators of energy metabolism (1Kahn B.B. 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Biochem. Soc. Trans. 2003; 31: 228-231Crossref PubMed Google Scholar). Its multiple downstream effects, including lowering of blood glucose levels, have identified AMPK as a promising target to treat type II diabetes mellitus and the metabolic syndrome (37Hardie D.G. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 185-210Crossref PubMed Scopus (356) Google Scholar). More detailed structural information about mammalian heterotrimeric AMPK is needed to understand its complex molecular architecture and function. Valuable but limited insight has been provided by x-ray structures of isolated AMPK domains: the catalytic domain of the human α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog Snf1 (38Rudolph M.J. Amodeo G.A. Bai Y. Tong L. Biochem. Biophys. Res. Commun. 2005; 337: 1224-1228Crossref PubMed Scopus (46) Google Scholar, 39Nayak V. Zhao K. Wyce A. Schwartz M.F. Lo W.S. Berger S.L. Marmorstein R. Structure. 2006; 14: 477-485Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), the glycogen-binding domain of the rat β-subunit (40Polekhina G. Feil S.C. Gupta A. O'Donnell P. Stapleton D. Parker M.W. Acta Crystallogr. Sect. F Struct. Biol. Crystallogr. Commun. 2005; 61: 39-42Crossref PubMed Scopus (12) Google Scholar), the Bateman domain of the γ-subunit yeast ortholog Snf4 (41Rudolph M.J. Amodeo G.A. Iram S.H. Hong S.P. Pirino G. Carlson M. Tong L. Structure. 2007; 15: 65-74Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and a CBS3-4-domain pair of the human γ-subunit (42Day P. Sharff A. Parra L. Cleasby A. Williams M. Horer S. Nar H. Redemann N. Tickle I. Yon J. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 587-596Crossref PubMed Scopus (76) Google Scholar). More informative, but still limited in their explanatory power, are the recent x-ray structures of truncated versions of a mammalian AMPK complex (14Xiao B. Heath R. Saiu P. Leiper F.C. Leone P. Jing C. Walker P.A. Haire L. Eccleston J.F. Davis C.T. Martin S.R. Carling D. Gamblin S.J. Nature. 2007; 449: 496-500Crossref PubMed Scopus (445) Google Scholar) and its yeast orthologs of Schizosaccharomyces pombe (43Townley R. Shapiro L. Science. 2007; 315: 1726-1729Crossref PubMed Scopus (153) Google Scholar) and Saccharomyces cerevisiae (44Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (132) Google Scholar) (supplemental Fig. S1). For successful crystallization, large truncations were introduced into the mammalian α- and β-subunits or their yeast homologs. The published core structures all lack the α-subunit kinase domain and a more or less large N-terminal part of the β-subunit. In the S. pombe and mammalian structures, almost 50% of the entire complex is missing. The S. cerevisiae structure (supplemental Fig. S1) is somewhat less truncated and contains a regulatory sequence (RS, α-subunit) and the glycogen-binding domain (β-subunit). A major contribution of these structures is the exact definition of subunit interactions. They confirm the structural consensus model of AMPK that had been challenged before (20Iseli T.J. Walter M. van Denderen B.J. Katsis F. Witters L.A. Kemp B.E. Michell B.J. Stapleton D. J. Biol. Chem. 2005; 280: 13395-13400Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21Wong K.A. Lodish H.F. J. Biol. Chem. 2006; 281: 36434-36442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In addition, the larger S. cerevisiae structure reveals novel interactions of the γ-subunit homologue with both the regulatory domain (α-subunit) and the glycogen-binding domain (β-subunit). The structures also identify the precise nucleotide binding sites on the γ-subunit (see also Fig. 9B). Although in yeast only a single AMP (or ATP) molecule is bound to the γ-homolog (43Townley R. Shapiro L. Science. 2007; 315: 1726-1729Crossref PubMed Scopus (153) Google Scholar, 44Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (132) Google Scholar), probably because yeast AMPK is not activated by AMP, the mammalian AMPK γ-subunit revealed three nucleotide binding sites (14Xiao B. Heath R. Saiu P. Leiper F.C. Leone P. Jing C. Walker P.A. Haire L. Eccleston J.F. Davis C.T. Martin S.R. Carling D. Gamblin S.J. Nature. 2007; 449: 496-500Crossref PubMed Scopus (445) Google Scholar). One site, corresponding to the yeast binding site, contains a non-exchangeable, fixed AMP, whereas the other two allow for ATP/AMP exchange and thus provide the AMP sensor function of AMPK. However, neither of the AMP-containing AMPK core structures showed an appreciable structural difference as compared with ATP-containing or nucleotide-free structures. This observation and the absence of the kinase domain in all known AMPK core structures have so far hampered a molecular explanation for AMP-dependent activation of the holo-complex. This mechanism necessarily involves a cross-talk between AMP binding on γ and the activating Thr172 phosphorylation on the α kinase domain, which most likely implies a conformational change and/or a domain movement. Therefore, detailed analysis of the full-length mammalian AMPK heterotrimer and its dynamic structure upon activation is obviously necessary. Here we report on the molecular shape of γ1-containing AMPK holoenzyme complexes obtained using different biophysical approaches. We have set up a high level expression and automated purification protocol of untagged AMPK protein on the basis of our His6-tagged tricistronic expression system (45Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expression Purif. 2003; 30: 230-237Crossref PubMed Scopus (118) Google Scholar). It yields sufficient quantities of homogeneous functional kinase complexes without the need of a potentially interfering purification tag. This allowed us to study AMPK in solution by small angle x-ray scattering (SAXS) and by different electron microscopy techniques, including single particle reconstruction. These data characterize AMPK as an elongated, flat particle with a large indentation and protrusions. Importantly, they demonstrate a ligand-induced conformational change of the AMPK heterotrimer upon binding of AMP. Plasmids and Expression of Proteins in Bacteria—Tricistronic AMPK expression plasmids were constructed as described earlier (45Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expression Purif. 2003; 30: 230-237Crossref PubMed Scopus (118) Google Scholar), but encoding non-tagged versions of the four different mammalian AMPK isoform combinationsα1β1γ1, α2β1γ1, α1β2γ1, and α2β2γ1 (GenBank™ accession numbers U40819, Z29486, X95577, AJ224538, and X95578). Proteins were expressed in Tuner (DE3) Escherichia coli cells (Novagen, EMD Chemicals Inc., Darmstadt, Germany). Expression of AMPK in rich medium in a self-constructed fermenter is described elsewhere (46Riek U. Tuerk R. Wallimann T. Schlattner U. Neumann D. Biotechniques. 2008; (in press)PubMed Google Scholar). Preculturing and expression in minimal medium was developed for full-length heterotrimeric AMPK and was also later successfully applied for the expression of the LKB1 complex as described recently (47Neumann D. Suter M. Tuerk R. Riek U. Wallimann T. Mol. Biotechnol. 2007; 36: 220-231Crossref PubMed Scopus (18) Google Scholar). The same method was used here for AMPK expression in a 42-liter bioreactor (MBR Switzerland), except that the growth temperature was kept at 34 °C, or in a 5-liter bioreactor (Minifors, Infors AG, Switzerland), except for some adaptations to the reduced vessel volume. After inoculation with cells from 400 ml of pre-culture, batch growth was continued for 12 h overnight at 32 °C with a stirrer speed at 1000 rpm and an air flow of about 4 liters/min until depletion of glucose and acetate (pO2 ∼100%). The feed was balanced with O2 consumption until reaching the pO2 limitation at the maximal air flow (∼7-8 liters/min; to obtain such high air flow, the original air inlet filter was changed to an AcroPac 300, Pall, Switzerland) and maximal stirrer speed (1250 rpm). Protein expression was induced with 50 mg/liter isopropyl 1-thio-β-d-galactopyranoside at around OD600 nm = 25 for 7.5 h at 34 °C. Bacteria were harvested by centrifugation, washed in physiological NaCl solution, and immediately frozen in liquid nitrogen. The yield per fermentation, expressed as wet weight of bacterial pellet, was reproducibly 200-250 or 1200-1400 g in the 5- or 42-liter bioreactor with 3 or 27 liters of medium, respectively. Protein Extraction—A 70-g aliquot of the frozen bacterial pellet was resuspended in lysis buffer (LysB: 30%(w/v) glycerol, 0.5 m sucrose, 50 mm HEPES, 2 mm MgCl2, pH 8.0, at 7 °C) to a total volume of about 200 ml. Cells were lysed with an Emulsiflex C5 high pressure homogenizer (Avestin, Germany), first applying 100-300 bar to resuspend cells, and then using 1200-1500 bar for cell lysis. The lysate was supplemented with 7 μl of Benzonase (purity grade II, Merck, Germany), stirred gently at 4 °C for 1 h, and centrifuged at 23,000 × g for 1 h to pellet cell debris. The clear supernatant was used for HPLC purification. Protein Purification—A new protocol was developed based on (i) the fact that untagged AMPK heterotrimer is able to bind to some metal affinity matrices, and on (ii) the availability of an Äkta Explorer 100 Air HPLC system (GE Healthcare) that was modified to allow automated multidimensional purification. Details of the machine setup will be published elsewhere. 5U. Riek, S. Ramirez, T. Wallimann, and U. Schlattner, unpublished data. The setup used here included the following columns: (i) XK 26/40 (GE Healthcare) containing 140 ml of Protino Ni-IDA (Macherey Nagel, Switzerland), (ii) XK 26/20 containing 45 ml with Reactive Red 120 fast flow highly cross-linked 6% agarose (R-6143 Sigma), (iii) two 1-ml Ni-HP columns (GE Healthcare), (iv) Superdex 200 16/60 (GE Healthcare); as well as a 10-ml Super-loop (SL) (GE Healthcare) at the first injection valve. The buffers used were: elution buffer (EluB: as LysB, but with the addition of 250 mm imidazole), Red Sepharose elution buffer (RSEluB: as LysB, but with the addition of 600 mm NaCl), Ni-HP elution buffer (HPEluB: as EluB but with the addition of 200 mm NaCl and 2 mm Tris(2-carboxyethyl)phosphine hydrochloride), size exclusion buffer one (SE1: 200 mm NaCl, 50 mm HEPES, 10 mm MgCl2, 8 mm EDTA, 2 mm Tris(2-carboxyethyl)phosphine hydrochloride, pH 8.0, at 7 °C), and size exclusion buffer two (SE2: as SE1, but without EDTA and with only 2 mm MgCl2). The fully automated purification procedure was carried out at 7 °C except for the Superdex column, which was run at 25 °C. Sample tubing at S1, S2, and S3 were connected to vessels containing LysB, EluB, and RSEluB, respectively, and were primed manually. The Ni-IDA column was equilibrated with LysB, the Reactive Red column with EluB, and the Ni-HP column with RSEluB. Tubing and valves were flushed with the appropriate buffers. Bacterial lysate was added to the sample vessel S1 and the automated run was started. Lysate was then applied to the Ni-IDA column at a flow rate of 4 ml/min using an air sensor to stop direct load injection. The column was washed with 160 ml of LysB (flow rate 5 ml/min at maximal 0.5 MPa pressure feedback). After flushing the system and P960 with EluB, bound proteins were eluted with EluB (5 ml/min, maximum 0.5 MPa). Elution fractions between 63 and 156 ml after EluB application were recovered in the S2 vessel and re-injected into the Reactive Red column at a flow rate of 1 ml/min, maximum 0.2 MPa. After washing with 94 ml of LysB at 2 ml/min (maximum 0.2 MPa), the volume eluting between 20 and 95 ml after application of RSEluB was collected in the S3 vessel and re-injected into the Ni-HP columns (1 ml/min, maximum 0.4 MPa). Columns were washed with 8 ml of LysB (0.5 ml/min, maximum 0.4 MPa) and eluted with EluB. A 5-ml peak fraction (starting at A > 280 0.6) was collected in the SL. The Superdex column was equilibrated with 200 ml of SE1 buffer (1.6 ml/min, maximum 0.1 MPa) and, finally, 4 ml of the SL fraction were loaded and 1-ml fractions were collected. Material left in the tubing and SL was collected later in separate fractions. For mass spectrometry (MS), STEM, transmission electron microscopy (TEM), SAXS, crystallization, and activity experiments, the first part of the size exclusion peak showing the highest specific AMPK activity was pooled, and subjected to a second size exclusion chromatography on the same Superdex column but using SE2 buffer without EDTA. Protein concentrations were determined using the Bradford Bio-Rad microassay (Bio-Rad) in SE2 buffer, calibrated by the 280-nm extinction coefficient for unfolded AMPK protein (α1β1γ1, 0.911; α2β2γ1, 0.892 g/liter). Dynamic Light Scattering—The dynamic light scattering signal was recorded on a DynaPro molecular sizing instrument (Wyatt). Samples after elution from the second size exclusion chromatography were concentrated to 10 mg/ml in SE2 buffer and centrifuged at 20,000 × g at 4 °C for 30 min prior to the measurements. Data were acquired using a 50-μl sample in an Eppendorf UVette cuvette at 20 °C with 10-s acquisition intervals and maximum laser intensity and analyzed with the software Dynamics. Enzyme Activity Assay—Following full activation by upstream kinases, activity of purified AMPK was determined by phosphorylation of the synthetic substrate SAMS in the presence of saturating AMP concentrations, using a non-radioactive, HPLC-based method that quantifies SAMS, phosphoSAMS, AMP, ADP, and ATP, as described previously (10Suter M. Riek U. Tuerk R. Schlattner U. Wallimann T. Neumann D. J. Biol. Chem. 2006; 281: 32207-32216Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Scanning Transmission Electron Microscopy (STEM)—Cross-linking of 1 mg/ml AMPK obtained in SE2 buffer after a second size exclusion chromatography was done at a final concentration of 1% glutaraldehyde (Fluka, Switzerland) for 60 s on ice and quenched with Tris, pH 8.0, at 4 °C. The cross-linked AMPK was then repurified by size exclusion on column 4 in SE2 to remove aggregates. An aliquot of this preparation was diluted 4× in SE2 buffer, and 5-μl aliquots were adsorbed for 60 s to glow-discharged STEM films (thin carbon films that span a thick fenestrated carbon layer covering 200-mesh/inch, gold-plated copper grids). The grids were blotted, washed on 5 drops of quartz double-distilled water, freeze-dried at -80 °C and 5 × 10-8 torr overnight in the microscope. Tobacco mosaic virus particles (kindly provided by R. Diaz Avalos, University of California, Davis, CA) were used for absolute mass calibration. These particles were similarly adsorbed to separate STEM films, washed on 4 drops of 10 mm ammonium acetate, and air-dried. A Vacuum Generators STEM HB-5 interfaced to a modular computer system (Tietz Video and Image Processing System GmbH, Gauting, Germany) was employed. Series of 512 × 512-pixel, dark-field images were recorded from the unstained sample at an acceleration voltage of 80 kV and a nominal magnification of ×200,000. The recording dose ranged from 474 to 945 electrons/nm2. The digital images were evaluated using the software package IMPSYS (48Müller S.A. Goldie K.N. Bürki R. Häring R. Engel A. Ultramicroscopy. 1992; 46: 317-334Crossref Scopus (142) Google Scholar). Accordingly, the projec
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