amp-activated protein kinase undergoes nucleotide ...bond lengths (Å) 0.005 0.003 bond angles ( )...

S1

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. ([email protected]).

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2319

S2

This file includes:

Supplementary Figures 1 to 5

Supplementary Tables 1 and 2

Supplementary Note

Supplementary References


S3

Supplementary Figure 1

a


S4

b

c

ATP-1

ATP-4

“ATP-3”Asp244

Asp89

Asp316

Arg170


S5

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.


S6


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


S7


Supplementary Figure 3. Effect of soaking ATP into co-crystallized AMP-bound

structure.

a

b


S8

(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.


S9


Supplementary Figure 4. Effect of soaking AMP into co-crystallized ATP-bound

structure.

a

b


S10

(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.


S11


a

b c

ATP-1

ATP-4 “ATP-3”

Asp244

Asp89

Asp316

Arg170

AMP-1

AMP-4 AMP-3

Asp244

Asp89

Asp316

Arg170


S12

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.


S13

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


S14

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.


S15

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.


S16

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).


amp-activated protein kinase undergoes nucleotide ...bond lengths (Å) 0.005 0.003 bond angles ( )...

Documents