amp-activated protein kinase undergoes nucleotide ...bond lengths (Å) 0.005 0.003 bond angles ( )...
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SUPPLEMENTARY INFORMATION
AMP-activated Protein Kinase undergoes
nucleotide-dependent conformational changes
Lei Chen1*, Jue Wang1, Yuan-Yuan Zhang1, S. Frank Yan2, Dietbert Neumann3, Uwe
Schlattner4,5, Zhi-Xin Wang1, and Jia-Wei Wu1
1MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences,
School of Life Sciences, Tsinghua University, Beijing 100084, China
2Molecular Design and Biostructure, Roche Pharma Research and Early Development
China, 720 Cai Lun Road, Building 5, Shanghai 201203, China
3Cardiovascular Research Institute Maastricht, Maastricht University, 6200 MD
Maastricht, The Netherlands
4INSERM U1055, Grenoble, France
5Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier, BP 53,
Grenoble, France
*Present address: Vollum Institute, Oregon Health & Science University.
Correspondence should be addressed. to J.W.W. (jiaweiwu@mail.tsinghua.edu.cn).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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This file includes:
Supplementary Figures 1 to 5
Supplementary Tables 1 and 2
Supplementary Note
Supplementary References
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 1
a
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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b
c
ATP-1
ATP-4
“ATP-3”Asp244
Asp89
Asp316
Arg170
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 1. Highly conserved conformation of two AMPK core proteins
co-crystallized with ATP.
(a) Sequence alignment of AMPK core. The code following each protein name is the
corresponding SwissProt ID. Secondary structure elements observed in the ATP-bound
structures are shown at the top of the alignment. The rat α1-subunit residues that are
deleted in the construct for crystallization are boxed. The hydrophobic residues on the
γ-subunit stabilizing the adenine rings of the AMP/ATP molecules are indicate by diamonds,
the hydrophilic residues interacting with the ribose moieties are indicated by triangles, and
the basic and polar residues coordinating the phosphate groups by asterisks above the
alignment. Residues at Sites 1, 3 and 4 are colored by red, green and blue, respectively.
(b) The co-crystallized ATP-bound structure of a chimeric AMPK core containing
Drosophila α (yellow), rat β1 (light green) and rat γ1 (light blue). The ATP molecules at
Sites 1 and 4 are highlighted in cyan sticks, and the Asp and Arg residues indicated in
Figure. 1a are shown in magenta and blue sticks, respectively. The inset shows the SA-omit
map (countered at 3.0 σ) for the ATP-bound structure of the chimeric AMPK core, which
clearly shows density for two ATP molecules at Sites 1 and 4. The simulated ATP molecule
at Site 3 was shown as gray stick, and the Tris molecule as green stick.
(c) Superposition of two ATP-bound AMPK core structures determined by
co-crystallization. The ATP-bound rat AMPK α1β1γ1 core is colored the same as that in
Figure. 1b. The assigned residues that are different between the rat β1- and human
β2-subunits are highlighted as red sticks.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 2
Supplementary Figure 2. Superposition of our ATP/AMP-bound mammalian AMPK
core structures with previous ATP/AMP-bound structures.
(a) Superposition of our ATP-bound AMPK core with previous ATP-bound structure. Our
co-crystallized ATP-bound structure is colored the same as that in Figure. 1b, and the ATP
molecules at Sites 1 and 4 are highlighted in cyan sticks. The Asp and Arg residues
indicated in Figure. 1a are shown in magenta and blue sticks, respectively. The previous
soaked ATP-bound structure (2V92)1 is shown in dark grey.
(b) Superposition of our AMP-bound AMPK core with previous AMP-bound structure. Our
AMP-bound structure is colored the same as that in Figure 1c, and the AMP molecules at
Sites 1, 3 and 4 are highlighted in yellow sticks. The previous AMP-bound structure
(2V8Q)1 is shown in grey.
a b
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 3
Supplementary Figure 3. Effect of soaking ATP into co-crystallized AMP-bound
structure.
a
b
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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(a) Schematic representation of the ATP-bound structure obtained by the soaking/replacing
method. The soaked ATP-bound structure is colored in deep teal, and the ATP and AMP
molecules are highlighted in cyan and yellow sticks, respectively. In this soaked ATP-bound
structure, ATP replaced two (Sites 1 and 3) out of the three molecules of AMP. The inset
shows the SA-omit map (countered at 3.0 σ) for the ATP molecule soaked into Site 3.
(b) Superposition of the soaked ATP-bound structure with the prototypical, co-crystallized
AMP-bound structure that is colored the same as that in Figure 1c. The soaked ATP-bound
structure retains nearly identical conformation to its prototype, except that Sites 1 and 3 are
now occupied by ATP.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 4
Supplementary Figure 4. Effect of soaking AMP into co-crystallized ATP-bound
structure.
a
b
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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(a) Schematic representation of the AMP-bound structure obtained by the soaking/replacing
method. The soaked AMP-bound structure is colored in marine and the AMP molecules are
indicated as yellow sticks. In the soaked AMP-bound structure, both ATP molecules at Sites
1 and 4 are substituted by AMP, but Site 3 is barely occupied by AMP due to the malformed
nucleotide-binding pocket retained from the prototypical ATP-bound structure (Fig. 2b and
2d). The inset shows the SA-omit map (countered at 3.0 σ) for a potential nucleotide (grey
line) at Site 3.
(b) Superposition of the soaked AMP-bound structure with the prototypical, co-crystallized
ATP-bound structure that is colored the same as that in Figure 1b. The soaked AMP-bound
structure retains nearly identical conformation to its prototype.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 5
a
b c
ATP-1
ATP-4 “ATP-3”
Asp244
Asp89
Asp316
Arg170
AMP-1
AMP-4 AMP-3
Asp244
Asp89
Asp316
Arg170
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Figure 5. Comparison of the co-crystal structures in complex with
ATP or AMP.
(a) Superposition of our ATP-bound and AMP-bound AMPK core structures. The structures
are shown in ribbon, and colored the same as that in Figure 1b and 1c, respectively. As
shown on the right, the 2Fo–Fc map for the ATP-bound AMPK core structure shows no
density for an ATP molecule at Site 3. The simulated ATP molecule at Site 3 was shown as
gray stick, and the Tris molecule as green stick.
(b) SA-omit map (countered at 3.0 σ) for the ATP-bound AMPK core structure clearly
shows density for two ATP molecules at Sites 1 and 4.
(c) SA-omit map (countered at 3.0 σ) for the AMP-bound AMPK core structure clearly
shows density for three AMP molecules at Sites 1, 3 and 4.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Table 1.
Data collection and refinement statistics for co-crystallized AMPK core structures.
co-crystallized ATP-bound AMPK Core (rat α1, β1 and γ1)
co-crystallized AMP-bound AMPK Core (rat α1, human β2 and rat γ1)
co-crystallized ATP-bound AMPK Core (Drosophila α, rat β1 and γ1)
Data collectiona Space group C2 P21212 C2221 Cell dimensions a, b, c (Å) 176.6, 40.5, 77.6 97.6, 115.3, 48.5 108.7, 151.3, 109.3
α, β, γ (°) 90, 105.1, 90 90, 90, 90 90, 90, 90
Resolution (Å) 50.0-2.5 (2.54-2.50)b 30.0-2.3 (2.34-2.30)b 30.0-2.7 (2.75-2.70)b Rmerge(%) 8.9 (58.1) 10.7 (47.7) 10.4 (35.8)
I / σI 13.1 (2.2) 10.9 (2.2) 8.6 (2.0) 178.0 (10.0)c
Completeness (%) 100.0 (100.0) 96.8 (91.5) 96.5 (83.6) 77.5 (15.1)c Redundancy 4.2 (4.1) 4.7 (3.3) 4.6 (2.9)
Refinement
Resolution (Å) 28.6-2.5 29.5-2.3 29.9-2.7 No. reflections 17922 23163 19448 Rwork / Rfree 23.7 / 25.4 19.4 / 25.2 20.8 / 25.2 No. atoms 3536 3715 Protein 3389 3544 3601 Ligand/ion 73 69 73 Water 74 102 81 B-factors (Average) 72.9 47.0 44.4 Protein 73.4 47.4 44.9 Ligand/ion 61.8 29.9 32.9 Water 62.6 42.2 31.1 R.m.s. deviations Bond lengths (Å) 0.009 0.004 0.009
Bond angles (°) 0.964 0.908 1.348 a Each data sets was collected from single crystal. b Values for highest resolution shell are shown in parenthesis. c These values indicates the statistics after anisotropic scaling and ellipsoidal truncation
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Table 2.
Data collection and refinement statistics for soaked AMPK core structures.
soaked ATP-bound AMPK Core (rat α1, human β2 and rat γ1)
soaked AMP-bound AMPK Core (rat α1, β1 and γ1)
Data collectiona Space group P21212 C2 Cell dimensions a, b, c (Å) 97.1, 116.0, 48.8 175.9, 40.5, 77.7
α, β, γ (°) 90, 90, 90 90, 105.5, 90
Resolution (Å) 30.0-2.6 (2.64-2.60)b 50.0-2.5 (2.54-2.50)b
Rmerge(%) 11.6 (39.7) 4.6 (35.6)
I / σI 12.9 (2.1) 22.5 (3.2)
Completeness (%) 98.7 (85.8) 99.6 (99.4) Redundancy 5.2 (2.9) 4.1 (4.0) Refinement Resolution (Å) 29.6-2.6 25.0-2.5 No. reflections 16423 17779 Rwork / Rfree 20.2 / 25.8 23.2 / 27.7 No. atoms 3683 3493 Protein 3544 3389 Ligand/ion 85 46 Water 54 58 B-factors (Average) 49.0 76.0 Protein 49.2 76.3 Ligand/ion 46.9 59.3 Water 40.2 76.1 R.m.s. deviations Bond lengths (Å) 0.005 0.003
Bond angles (°) 1.023 0.729 a Both data sets were collected from single crystal. b Values for highest resolution shell are shown in parenthesis.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary Note
All diffraction data sets were processed using the HKL20002. The structures were solved by
molecular replacement using Phaser3 with the mammalian AMPK core in complex with
AMP (2V8Q) as search model1. Standard refinement was performed with the programs
Phenix4 and Coot5. PROCHECK6 indicated that none of the residues in the structures is in
the disallowed region of the Ramachandran plot.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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Supplementary References
1. Xiao, B. et al. Structural basis for AMP binding to mammalian AMP-activated
protein kinase. Nature 449, 496-500 (2007).
2. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Macromolecular Crystallography, Pt A 276, 307-326 (1997).
3. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40,
658-674 (2007).
4. Adams, P.D. et al. PHENIX: building new software for automated crystallographic
structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948-54 (2002).
5. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
Crystallogr. D Biol. Crystallogr. 60, 2126-32 (2004).
6. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. Procheck - a
Program to Check the Stereochemical Quality of Protein Structures. Journal of
Applied Crystallography 26, 283-291 (1993).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319
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