Figures and data in Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6

Figures
Videos
Tables

7 figures, 1 video and 3 tables

Figures

Figure 1

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Inhibitory activities of pS9 and LRP6 a-motif peptides.

(A and B) ³²P incorporation into the peIF2b substrate over time for GSK-3β 26-383, inhibited by the S9 peptide (K_i = 60 ± 11 μM) (A) or the LRP6 a-motif peptide (K_i = 1.4 ± 0.2 μM). The number of replicates is either two or four for each inhibitor concentration, except for the 15 µM LrpA peptide timecourse, where there is only one measurement. (C) Determination of steady-state kinetic constants for GSK-3β 26-383, GSK-3β 26-383^F93A, GSK-3β 26-383^F93G, GSK-3β 1-383, and GSK-3β 1-383^pS9. Error bars represent the standard error of the mean. The number of replicates for each concentration is between 2 and 4 for all the concentration points, except for the 1 µM point of GSK-3 26-383, the 80 µM point of GSK-3β 26-383^F93A, and the 50 and 80 µM points of GSK-3β 26-383^F93G, which have only one measurement.

https://doi.org/10.7554/eLife.01998.003

Figure 2

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Inhibitory peptide binding to GSK-3.

(A) Overall structure of GSK-3 bound to inhibitory peptides. The superimposed LRP6 c-motif (pink sticks), e-motif (light green sticks) and pS9 auto-inhibitory N-terminal peptide (light blue sticks) bind to the same substrate-binding pocket between the C-loop (yellow) and activation loop (red). A molecule of ADP binds to the deep cleft located between the N-terminal (white) and C-terminal (grey) lobes, and the Axin helix (purple) binds at the C-lobe. The glycine-rich loop (cyan) and αC-helix are also indicated. The inset shows the protein sequences of the peptide residues that are visible in the structures. The P+4 phosphorylated residues are indicated in orange. The loop between the N-terminal peptides and the first β strand of the N-terminal lobe is partially disordered (dotted line). Oxygen atoms are shown in red, nitrogen in blue, phosphorus in orange, and sulfur in yellow. (B) Surface representation of the substrate-binding pocket between the C-loop (yellow) and activation loop (red) of GSK-3. The inhibitory peptides, pS9 auto-inhibitory N-terminal peptide (light blue sticks), LRP6 c-motif (pink sticks) and e-motif (light green sticks) are superimposed, and the residues of the peptides are labeled according to the primed substrate numbering, with the phospho-serine or threonine at the P+4 position. Side chains of GSK-3 residues F93, Y216 and I217, which interact with the peptides, are also depicted as sticks. (C) Peptide inhibitor-free structure near the C-loop and activation loop. A molecule of glycerol is bound to three basic residues that interact with the phosphate at the substrate P+4 site. Hydrogen bonds are shown as dashed lines. (D–F) Interactions between GSK-3 and inhibitory peptides. The structural water molecules that interact between the carbonyl groups of Y216 and the P+1 proline residues of LRP6 c-motif and e-motif peptides are depicted as red spheres.

https://doi.org/10.7554/eLife.01998.006

Figure 3

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Conformational changes in GSK-3 upon inhibitor peptide binding.

(A) Superposition of α-carbon traces of GSK-3 structures. The maximum deviation between the structures is at the C-loop region, which is color coded according to the key at the bottom of the panel. The dashed line indicates the maximum distance (8.5 Å) between the α-carbon of residue Phe93 seen in peptide substrate-bound and peptide-free structures. The activation loops (red) align very well, indicating that there is no conformational change at the activation loop upon peptide substrate binding. (B) Changes of the C-loop upon peptide substrate binding. Superposition of the peptide substrate-free GSK-3 (inhibitor-free; Table 2) (orange) and the GSK-3/pS9 peptide (top panel), LRP6 c-motif (middle panel), or LRP6 e-motif (bottom panel) complexes. Hydrogen bonds between the backbones of the P+4 and P+5 residues of the peptides and the backbone of the C-loop from R92 to K94 are shown as dashed lines; only the main-chain atoms of R92 and K94 are depicted. The change in the C-loop upon peptide binding alters the positions of the preceding β strands (broad arrows). R96 adopts a conformation to accommodate the P+4 substrate phosphate. (C) Superposition of GSK-3 and active PKA structures. The catalytic residues, including K85, E97 and the DFG motif, of the peptide-free GSK-3 (orange), peptide-free GSK-3 with phosphorylated Y216 (purple) or peptide-bound GSK-3 (light blue) adopt similar conformations as those of the active PKA structures (gold/brown). The double-headed arrow indicates the shorter length of the αC-helix of GSK-3 relative to that of PKA. The hydrogen bond between phosphorylated T197 (pT197) and H87 is shown as dashed line; only H87 of the PKA/peptide complex is depicted for clarity. pT197 of PKA places a phosphate group in the space equivalent to that occupied by the substrate phosphate in GSK-3.

https://doi.org/10.7554/eLife.01998.007

Figure 4

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Conformational change of the glycine-rich loop upon inhibitor peptide binding.

(A) *Left panel*: superposition of α-carbon traces of five selected GSK-3 structures. The dashed line indicates the maximum deviation at the glycine-rich loops among the currently solved GSK-3 structures (4 Å at the α-carbon of residue S66 between the peptide-free (orange/purple) and the peptide-bound (light blue) GSK-3). Some GSK-3 structures show an intermediate position of the glycine-rich loop (green/pink) due to interaction with another GSK-3 molecule in the crystal lattice (not shown in the figure for clarity, please refer to PDB 1H8F/1PYX). The inset shows the close-up of the glycine-rich loops of the five selected GSK-3 structures. Upon peptide binding, F67 packs against the T7 or P1570/P1605 at the P+2 residue of the pS9 or LRP6 peptides. *Right panel*: superposition of α-carbon traces of three selected PKA structures. The glycine-rich loop of the nucleotide-free PKA (light purple) is at the ‘highest’ position, due to the open conformation of the inactive kinase structure. In the nucleotide-bound state (gold), the glycine-rich loop moves toward the C-terminal lobe, and it moves further down by 4 Å upon binding to a peptide in its substrate-binding site (brown). The inset shows the close-up of the glycine-rich loops of the three selected PKA structures. The equivalent residues of GSK-3 S66 and F67, PKA S53 and F54, are depicted as sticks. The ADP and AlF₃ molecules are also shown. (B) Close-up comparisons of the glycine-rich loops in the nucleotide-bound state (upper panel) and nucleotide/peptide-bound transition state (lower panel) of GSK-3 and PKA. (C) Comparisons of the electron density (gray mesh, 2Fo-Fc map contoured at 0.8 σ) of the glycine-rich loops in the ADP-bound state (upper panel) and ADP–AlF₃ transition state (lower panel). The upper and lower conformations of the glycine-rich loops are indicated. GSK-3 S66 and F67 are depicted as sticks, and the ADP and AlF₃ molecules are also shown.

https://doi.org/10.7554/eLife.01998.008

Figure 5

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Comparison of GSK-3 inhibitory peptide orientation with other kinase: peptide complexes.

Superposition of the GSK-3/LRP6 c-motif structure with the (A) PKA:PKI peptide (PDB 1L3R); (B) CDK2:p107 peptide (PDB 1QMZ); (C) AKT:GSK-3 peptide (PDB 3CQU); and (D) DYRK:consensus peptide (PDB 2WO6) substrate complexes. The β-phosphate of the ADP molecule in the catalytic site of GSK-3 is shown at the top of each panel, with phosphorus in orange and oxygen in red.

https://doi.org/10.7554/eLife.01998.010

Figure 6

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GSK-3 sequence alignments near the C-loop.

White letters on black background indicates identical residues, and bold letters denote similar residues. Secondary structure elements are shown above the alignment. The C-loop is marked in the red box, with Phe93 indicated by the asterisk.

https://doi.org/10.7554/eLife.01998.011

Figure 7

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GSK-3 substrate specificity.

(A) Amino acid frequency logo diagram for each of the positions P+0 through P+5 for the 75 GSK-3 substrates listed in Table 3. The diagram was created using WebLogo (Crooks et al., 2004). (B) Conformational change of Y216 upon peptide binding. In the absence of peptide and Y216 phosphorylation, the ring of Y216 packs against V214. In the presence of peptide, Y216 points outward, away from the peptide, and adopts an identical conformation to that seen when it is phosphorylated (purple, GSK-3/Axin/ADZ structure; PDB 1O9U [Dajani et al., 2003]). The ring of Y216 packs against the P+1 residue in each of the peptide complexes. (C) Model of basic P+1 residue interaction with phosphorylated Y216. The phosphate group of phosphorylated Y216 could interact with the positively charged moiety of arginine at P+1 of the peptide substrate. As seen in PDB 1O9U, Arg220 and Arg223 would also chelate the phosphate group. Also shown are peptide-binding residues on the glycine-rich loop (cyan), C-loop (yellow) and activation loop (red).

https://doi.org/10.7554/eLife.01998.013

Videos

Video 1

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posterframe for video — Conformational changes in GSK-3 associated with peptide substrate binding.

The video shows the ADP and Axin-bound enzyme changing between the inhibitor-free and pS9-bound states. Key structural elements are colored as in Figure 2A.

https://doi.org/10.7554/eLife.01998.009

Tables

Table 1

Kinetic parameters for GSK-3β variants

https://doi.org/10.7554/eLife.01998.004

	K_M (µM)	k_cat (s⁻¹, × 10⁻²)	k_cat/k_M (M⁻¹•s⁻¹, × 10³)
GSK-3β 26-383	8.5 ± 3.8	7.9 ± 1.2	9.3 ± 4.4
GSK-3β 26-383^F93A	39.1 ± 13.8	5.1 ± 0.6	1.3 ± 0.5
GSK-3β 26-383^F93G	21.3 ± 14.2	2.3 ± 0.4	1.1 ± 0.8
GSK-3β 1-383	27.8 ± 15.7	15.6 ± 3.6	5.6 ± 3.4
GSK-3β 1-383^pS9	21.7 ± 9.0	2.7 ± 0.4	1.2 ± 0.5

Table 2

X-ray crystallography data collection and refinement statistics

https://doi.org/10.7554/eLife.01998.005

	Inhibitor-free	pS9	pS9-AlF₃	LRP6 c-motif	LRP6 e-motif
PDB code	4NM0	4NM3	4NU1	4NM5	4NM7
Data collection*
Space group	P6₁22	P6₁22	P6₁22	P6₁22	P6₁22
Unit cell lengths a, c (Å)	81.3, 280.8	81.0, 281.1	81.0, 280.5	81.7, 280.9	82.0, 280.3
Beamline	SSRL 11-1	SSRL 11-1	APS 23-ID-B	SSRL 12-2	SSRL 12-2
Wavelength (Å)	1.033	1.033	1.033	1.00	1.00
Resolution range (Å)	39.1–2.50	39.0–2.10	46.8–2.50	39.2–2.30	39.4–2.30
(last shell)	(2.60–2.50)	(2.16–2.10)	(2.60–2.50)	(2.38–2.30)	(2.38–2.30)
Unique reflections	20,069	33,093	19,922	25,685	25,932
CC_1/2†	0.999 (0.726)	1.00 (0.673)	0.999 (0.814)	0.999 (0.459)	1.00 (0.485)
R_merge‡	0.106 (1.74)	0.100 (4.88)	0.303 (6.772)	0.141 (3.63)	0.136 (4.31)
<I>/<σI>	18.4 (1.2)	24.5 (0.8)	13.0 (0.7)	18.9 (1.0)	17.8 (0.9)
Completeness (%)	100 (99.9)	99.9 (99.7)	100 (100)	99.7 (99.6)	100 (100)
Multiplicity	9.6 (9.9)	18.6 (17.9)	21.0 (21.6)	18.9 (18.1)	18.7 (18.2)
Refinement
No. reflections work/test set	19,958/979	32,924/1658	19,823/974	25,608/1283	25,838/1292
R_work/R_free§	0.191/0.240	0.194/0.242	0.191/0.245	0.183/0.232	0.183/0.234
Number of atoms
GSK-3	2780	2885	2862	2850	2858
Axin	165	153	149	155	155
LrpC/E peptide	–	–	–	44	44
ADP	27	27	27	27	27
Mg²⁺	2	2	2	2	2
Cl⁻	1	1	–	1	1
Glycerol	30	24	12	24	18
DTT	8	8	–	–	8
AlF₃	–	–	4	–	–
NO₃⁻	–	–	4	–	–
Water	164	197	98	121	106
B-factors (Å²)
GSK-3	52.6	55.0	75.1	69.7	69.7
Axin	59.5	54.0	78.1	75.2	77.8
LrpC/E peptide	–	–	–	101	100
ADP	65.3	50.3	57.5	72.2	56.5
Mg²⁺	88.5	53.8	57.6	86.4	69.6
Cl⁻	77.1	71.1	–	90.9	96.8
Glycerol	65.2	79.4	93.1	94.6	97.2
DTT	99.3	102	–	–	131
AlF₃	–	–	83.6	–	–
NO₃⁻	–	–	100.8	–	–
Water	46.4	52.3	61.1	64.3	64.6
Rmsd
Bond lengths (Å)	0.003	0.005	0.003	0.002	0.004
Bond angles (°)	0.63	0.91	0.63	0.63	0.76
Ramachandran plot (%)¶
Favored regions	96.5	96.9	96.1	95.6	95.8
Additional allowed regions	3.5	3.1	3.9	3.9	4.2
Outliers	0	0	0.5	0.5	0

*

Values in parentheses are for highest-resolution shell. Rmsd, root mean square deviation.
†

As defined in Aimless (Evans and Murshudov, 2013).
‡

R_merge = Σ_hΣ_I|I_I(h)–< I(h) > |/Σ_hΣ_I(h), where I_I(h) is the I^th measurement of reflection h, and < I(h) > is the weighted mean of all measurements of h.
§

R = Σ_h|F_obs(h)–F_calc(h)|/Σ_h|F_obs(h)|. R_work and R_free were calculated using the working and test reflection sets, respectively.
¶

As defined in MolProbity (Chen et al., 2010).

Table 3

Confirmed GSK-3 biological targets (adapted from Sutherland, 2011)

https://doi.org/10.7554/eLife.01998.012

Protein	P+0 residue	P+0 to P+5 sequence	UniprotKB #
APC	1501	SCSSSL	(P25054)
ATP-citrate lyase	447	TPAPSR	(P53396)
ATP-citrate lyase	451	SRTASF	(P53396)
Axin	317	SANDSE	(O15169)
Axin	321	SEQQSL	(O15169)
BCL-3	398	SPSSSP	(P20749)
BCL-3	402	SPSQSP	(P20749)
β-catenin	33	SGIHSG	(P35222)
	37	SGATTT
	41	TTAPSL
C/EBPα	226	TPPPTP	(P49715)
C/EBPα	230	TPVPSP	(P49715)
C/EBPβ	223	SLSTSS	(P17676)
	227	SSSSSP
	231	SPPGTP
Ci155 (Gli3)	861	SRRSSG	(P10071)
	873	SRRSSE
	903	SRRSSE
CLASP2	533	SRESSR	(O71522)
CLASP2	537	SRDTSP	(O71522)
CRMP2	514	TPASSA	(Q16555)
CRMP2	518	SAKTSP	(Q16555)
CRMP4	514	TPAGSA	(Q14195)
CRMP4	518	SARGSP	(Q14195)
CREB	129	SRRPSY	(P16220)
CRY2	554	SGPASP	(Q49AN0)
Cytidine triphosphate synthetase	571	SGSSSP	(P17812)
Dynamin I	776	TSSPTP	(Q05193)
eIF2B	540	SRGGSP	(Q13144)
FAK	722	SPRSSE	(Q05397)
Glycogen Synthase	641	SVPPSP	(P13807)
	645	SPSLSR
	649	SRHSSP
	653	SPHQSE
Heat shock factor 1	303	SPPQSP	(Q00613)
HIF1α	551	STQDTD	(Q16665)
hnRNP D	83	SPRHSE	(Q14103)
IRS1	337	SRPASV	(P35568)
c-jun	239	TPPLSP	(P05412)
MAP1B	1396	SPLRSP	(P46821)
Mcl1	159	SLPSTP	(Q07820)
Mdm2	242	SDQFSV	(Q00987)
Mdm2	256	SEDYSL	(Q00987)
MLK3	789	SPLPSP	(Q16584)
c-myc	58	TPPLSP	(P01106)
Myocardin	451	STSSSP	(Q8IZQ8)
	455	SPPISP
	459	SPASSD
	626	STFLSP
	630	SPQCSP
	634	SPQHSP
NDRG1	342	SRSHTS	(Q92597)
p130Rb	948	SHQNSP	(Q08999)
	962	SRDSSP
	982	SAPPTP
p53	33	SPLPSQ	(P04637)
PITK	1007	SKTVSF	(O15084)
Polycystin-2	76	SPPLSS	(Q13563)
Presenilin-1	397	SATASG	(P49768)
Presenilin-1	353	STPESR	(P49768)
PP1 G-subunit	38	SPQPSR
PP1 G-subunit	42	SRRGSE
PTEN	362	STSVTP	(P60484)
PTEN	366	TPVDSD	(P60484)
Snail	96	SGKGSQ	(O95863)
	100	SQPPSP
SREBP1a	426	TPPPSD	(P36956)
SREBP1a	430	SDAGSP	(P36956)
Tau	525	SRSRTP	(P10693)
	548	TPPKSP
	713	SPVVSG
	717	SGDTSP
TSC2	1379	SQPLSK	(P49815)
TSC2	1383	SKSSSS	(P49815)
VDAC	51	TTKVTG	(P21796)
von Hippel-Lindau	68	SREPSQ	(P40337)

Numbers listed are residue numbers of the P+0 residue of the human proteins, with sequences representing the P+0 through P+5 residues for each target sequence. UniprotKB accession numbers are listed to the right.