Biochemical and Biophysical Research Communications 504 (2018) 519e524

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Biochemical and Biophysical Research Communications

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Crystal structure of GSK3b in complex with the flavonoid, morin

** Kuglae Kim a, c, Jeong Seok Cha a, Jin-Sik Kim b, d, Jinsook Ahn b, Nam-Chul Ha b, , * Hyun-Soo Cho a, a Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea b Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea c Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27516, USA d Unit on Structural and Chemical Biology of Membrane Proteins, Cell Biology and Neurobiology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 35A Convent Drive, Bethesda, MD 20892, USA article info abstract

Article history: GSK3b is a key that plays a role in cellular signaling pathways. In Alzheimer's disease (AD), GSK3b Received 21 August 2018 has been implicated in hyperphosphorylation of tau proteins in the neuron, which is a hallmark of AD. Accepted 28 August 2018 Morin, a flavonoid that is abundant in nature, was found as an inhibitor of GSK3b that can reduce tau Available online 7 September 2018 pathology in vivo and in vitro. In this study, we determined the crystal structure of GSK3b in complex with morin. The structure revealed that morin inhibits GSK3b by binding to the ATP binding pocket. Our Keywords: findings augment the potential of morin as a functional food to help prevent AD, as well as to provide GSK3 structural information to develop new therapeutics based on the morin skeleton. Morin © Alzheimer's disease 2018 Elsevier Inc. All rights reserved. Complex structure Natural compound

1. Introduction microtubules and formation of filamentous self-aggregates in the neuron [6]. This insoluble deposition of tau proteins in neurons Glycogen synthase kinase 3b (GSK3b) is a Ser/Thr protein kinase results in a class of diseases collectively known as tauopathies [7]. that was originally identified as a key regulator of glycogen meta- Tauopathies encompass more than 20 clinicopathological entities, bolism in the insulin signaling pathway. The is now known including Alzheimer's disease (AD). b-Amyloid precursor protein to be involved in various biological functions, including the Wnt, (APP), presenilin, and tau exert crucial roles in the pathogenesis of Hedgehog, Notch, and inflammation signaling pathways [1,2]. Un- AD. Increased production of amyloid b protein (Ab), converted from like most , GSK3b has a constitutive activity in its resting APP by presenilin, results in deposition into plaque in the extra- state and transduces cellular signaling via inhibition of its activity cellular region, and leads to the pathogenic hyperphosphorylation [3]. of tau by increasing the activity of GSK3b. However, how GSK3b In neurons, GSK3b is highly expressed and plays important roles activity is elevated by Ab has not been fully understood [6]. in regulating structural and metabolic plasticity by phosphory- Morin (3,5,7,20,40-pentahydroxyflavone) is a flavonoid with lating diverse proteins [4]. Especially, GSK3b phosphorylates tau antioxidant properties that can protect cells against damage from proteins, which are microtubule-associated proteins abundantly oxygen radicals. Morin was originally isolated from mulberry figs expressed in neurons of the peripheral and central nervous systems andheartwoodofoldfustic(Chlorophora tinctoria), and it is rich in [5]. The phosphorylation state of tau regulates the binding ability many oriental medicinal herbs [8]; thus, it can be isolated from and stabilization of microtubules. However, the hyper- nature. Morin has diverse physiological effects, including anti- phosphorylation of tau proteins can lead to their detachment from cancer effects [9,10]. Morin was previously reported to directly inhibit GSK3b, resulting in blocking GSK3b-induced tau phos- phorylation in vitro and in vivo [11]. Furthermore, morin attenu- ated Ab-induced tau phosphorylation and cytotoxicity in human * Corresponding author. neurons [11]. Reductions in tau hyperphosphorylation were ** Corresponding author. observed in hippocampal neurons in a 3xTg-AD mouse model by E-mail addresses: [email protected] (N.-C. Ha), [email protected] (H.-S. Cho). intraperitoneal administration of morin for 7 days [11]. However, https://doi.org/10.1016/j.bbrc.2018.08.182 0006-291X/© 2018 Elsevier Inc. All rights reserved. 520 K. Kim et al. / Biochemical and Biophysical Research Communications 504 (2018) 519e524 it remains unknown how morin inhibits GSK3b at the atomic 2.4. Surface plasmon resonance (SPR) level. In this study, we present the structural basis for the role of morin by presenting the crystal structure of GSK3b in complex For the SPR experiment, we prepared the His6-tagged GSK3b with morin. protein by skipping the TEV cleavage step during purification. The purified His6-tagged GSK3b protein was immobilized in the GLH sensor chip (BIO-RAD) by using the ProteOn™ XPR36 machine. 2. Materials and methods Morin in DMSO was prepared at various concentrations (0, 6.25, 12.5, 25, 50, and 100 mM), and each was passed over the GSK3b- fi 2.1. Expression and puri cation immobilized chip. All data were processed by the 1:1 Langmuir binding model, and the binding kinetics and dissociation constant fi The expression and puri cation of the mouse GSK3b kinase (kD) were calculated. domain have been previously described [12]. Mouse GSK3b (resi- e dues 14 420) was ligated into the pFASTBAC-HTA vector (Invi- 3. Results trogen, USA) to generate the recombinant baculovirus for the Bac- to-Bac baculovirus expression system (Invitrogen). The resulting 3.1. Structural determination and overall structure construct had an appended His6-affinity purification tag and a cleavage site for the TEV protease to GSK3b sequence. According to Recombinant GSK3b produced in the insect cells using the the manufacturer's instructions, recombinant baculovirus was baculovirus expression system was co-crystallized with morin. The constructed. The protein was expressed after infection of the virus crystal structure of the complex was solved by the molecular 6 at a multiplicity of infection of 5 in 2.0 10 SF9 cells in CCM3 replacement method using a known structure of human GSK3b [15] media (Hyclone, USA). The cells were harvested by centrifugation as a search model. The asymmetric unit contained four GSK3b and stored at 80 C until use. The cells were thawed in a buffer protomers. The Ramachandran plot showed that 100% of the non- consisting of 20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imid- glycine residues were in favored or allowed regions. The refined azole, 3 mM b-mercaptoethanol, and 10% glycerol and disrupted by model presented good stereochemistry, as evaluated by the pro- homogenization (100 strokes). The cell lysate was applied to Ni- gram MolProbity [19]. The crystallographic data and refinement fi NTA metal af nity chromatography, and then, the His6-tagged statistics are shown in Table 1. protein was pooled and incubated with the recombinant TEV pro- The overall structures of GSK3b was almost identical to the tease overnight to cleave the His6 tag. After changing the buffer by a GSK3b structures of apo-form, ADP complex, ADP, and an inhibitory desalting column in a buffer containing 20 mM HEPES (pH 7.5), peptide bound complex [20]. Each protomer consisted of the N- 50 mM NaCl, and 1 mM DTT, Resource S column (GE Healthcare, terminal domain (N-lobe) and the C-terminal domain (C-lobe) e USA) was applied using a linear gradient of 0.05 1 M NaCl in between the nucleotide and substrate binding sites as shown in 20 mM HEPES (pH 7.5). The GSK3b protein was eluted at 150 mM Fig. 1A. All protomers contained a phosphorylated tyrosine at res- fi NaCl, and the fractions were pooled. The nal protein sample was idue 216 as observed in previous structures [20]. The asymmetric concentrated to 5 mg/mL using a centrifugal concentrator (Milli- unit could be divided into two dimers, and the root-mean-square pore, USA). deviation (RMSD) value between the two dimers was 2.68 Å be- tween the 637 matched Ca atoms (Fig. 1B). Each dimer consists of 2.2. Crystallization two GSK3b molecules that had interactions between the N-lobe and C-lobe (Fig. 1C), which was different from the putative dimeric Prior to the crystallization, morin (Sigma, USA) was incubated at 5mM final concentration in the GSK3b protein sample (5 mg/ml) Table 1 for 30 min at 4 C. Co-crystallization of the GSK3b protein with Data collection and refinement statistics. morin was performed using the hanging drop vapor diffusion GSK3b morin complex technique. The plate crystals were observed at 17 C under a con- dition optimized for hanging drop experiments by mixing 1.5 mLof Data collection Space group P2 the protein sample with the same volume of the reservoir solution 1 Cell dimensions containing 18% (v/v) PEG 4000, 100 mM sodium citrate (pH 6.5), a, b, c (Å) 67.6, 134.4, 100.4 and 5% (v/v) 2-propanol. b () 103.8 Resolution (Å) 40.0e2.14 (2.18e2.14)

Rmerge (%) 7.6 (34.9) s 2.3. Data collection and structural determination I/ I 38.2 (3.8) Completeness (%) 97.8 (95.0) fl Redundancy 6.0 (4.3) The crystals were brie y soaked in a cryoprotectant solution of Refinement the reservoir solution supplemented with 20% ethylene glycol. Resolution (Å) 40.00e2.14 Diffraction data were collected from single crystals flash-frozen No. reflections 88,879 a at 173 C in a nitrogen stream. The dataset was measured to a Rwork/Rfree (%) 19.1/22.6 No. atoms 2.14 Å resolution using a beamline 5C on a Pohang accelerator Protein 10,463 laboratory synchrotron with X-rays at a 1.0 Å wavelength [13]. The Ligand/ion 68 data were auto-indexed and processed with the HKL suite [14]. The Water 422 crystals of GSK3b with morin complex belonged to the space group B-factors Protein 49.29 P21. The crystal structure was solved by molecular replacement Ligand/ion 49.71 using human GSK3b, PDB code 1I09 [15] as the search model in the Water 43.99 program MOLREP [16]. Coot was used for the visualization of the R.M.S deviations Bond length 0.020 electron density maps and the manual rebuilding of the atomic Bond angle 1.62 fi model [17]. The model was re ned using the program REFMAC5 The numbers in parentheses are statistics for the highest-resolution shell. a [18] at a 2.14 Å resolution. Rfree was calculated with 5% of the data set. K. Kim et al. / Biochemical and Biophysical Research Communications 504 (2018) 519e524 521

Fig. 1. Overall structure of GSK3b. (A) The dimeric structure of GSK3b/morin is shown in the cartoon representation. Each protomer is colored differently (light blue and cyan). The morin molecules and phos- phorylated Tyr residues are shown in stick representations. (B) The superimposed structure of dimer group 1 (light blue and cyan for chains A and B, respectively) and group 2 (magenta and yellow for chains C and D, respectively) is shown in the ribbon diagram. (C) The superimposed structure of GSK3b/morin (light blue and cyan) and the putative dimeric unit previously observed (PDB code: 1H8F) (light blue and salmon) is shown in the cartoon diagram. (D) Close-up view of the dimeric interface between Gly-rich loop of protomer 1 (light blue) and C-lobe of protomer 2 (cyan), marked by a black box. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) unit of human and mouse GSK3b [20]. The Gly-rich loop in the involved in the hydrophobic interaction with morin A-C rings. The nucleotide- in the pocket was additionally hydroxyl group at C-7 of the morin A ring makes the hydrogen lined with Ser261 and Gly262 residues from the C-lobe of the other bonds with the Val135 backbone amide group and Asp133 back- protomer in each dimeric group (Fig. 1D). bone carbonyl group (Fig. 2C). The hydroxyl group at the C-3 in the C ring makes a polar interaction with the side chain of Lys85. The dihydroxybenzene group of the B ring occupied the nucleotide- 3.2. Binding of morin binding site mainly via the hydrophobic interaction. Only a hydrogen bond with the backbone carbonyl group of Asn64 was The electron density for morin molecules was clear and strong found in the B ring. Interestingly, the hydroxyl group at C-4' of the B enough to identify the molecules in the structure (Fig. 2A and B). ring also formed a hydrogen bond with the backbone amide group The two protomers (chains A and B) of GSK3b in the asymmetric of Gly262 in the other protomer in the dimeric unit, which unit contained morin molecules in the active site pockets. Flavo- appeared to reinforce the dimeric arrangement shown in the noids have the general structure of a 15-carbon skeleton, consisting crystal structure (Fig. 2D). of two phenyl rings (A and B) and heterocyclic ring (C). The planar A and C rings were combined, and the B ring was connected to the C2 in the C ring. The B ring of morin was called the resorcinol or meta- 3.3. Structural comparison of morin binding dihydroxybenzene group. The B ring was linked to the A-C ring of morin at C-2 with a slight tilting angle (~10) in the map. The structural superposition onto the GSK3b structure in com- þ Morin occupied the hydrophobic pocket for the adenine- plex with ADP-Mg2 and a inhibitory peptide (PDB code: 4NM3, binding site, lined with three b strands of the N-lobe, the con- RMSD 0.624 A between 291 Ca atoms) revealed differential binding þ necting loop between the N- and C-lobes, and two b-strands of the between morin and ADP-Mg2 [3]. The A and C rings of morin were C-lobe. The Gly-rich loop is known for nucleotide binding. Val110 structurally comparable to the adenine moiety of ADP, and from the connecting loop between the N- and C-lobes, Leu188 from exhibited deeper binding than the adenine moiety (Fig. 3A). a b-strand of the C-lobe, and Cys199 from the activation loop were However, the amine group of N-1 in adenine and the hydroxyl 522 K. Kim et al. / Biochemical and Biophysical Research Communications 504 (2018) 519e524

Fig. 2. The morin binding environment. (A) The protomer of GSK3b (cartoon diagram) in complex with morin (stick representation; yellow and red). The N-lobe is in green, and the C-lobe in light blue. The ATP binding pocket is marked by a red broken box. (B) The electron density map of morin and the vicinity residues (green and light blue) are shown in mesh. The 2Fo-Fc map of morin and residues are contoured at 2.0 s and are shown in the grey mesh. (C) The detailed view of the binding mode of morin to GSK3b is shown in stick diagram. Hydrogen bonds are indicated by the black dashed lines. The names of the rings and the carbon numbers of morin are labeled in red. (D) Closed up view of the interaction of morin (yellow) and G262 of protomer 2 (cyan) are shown in the stick diagram. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

þ Fig. 3. Structural comparison of binding of morin to ADP-Mg2 in GSK3b, and to morin in DAPK1. þ þ (A) Superposed GSK3b structures in complex with morin (yellow backbone) and ADP-Mg2 (magenta backbone). N, O, and Mg2 are colored by blue, red, and green, respectively. The GSK3b is shown in a Ca representation. (B) The superposed structure of GSK3b/morin complex and DAPK1/morin (PDB; 5AUY) complex. The colors indicate the half circle þ symbols and labeled. N, O, and Mg2 are colored by blue, red, and green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) group of C-7 in the morin A ring were very close and commonly atom in the a-phosphate that forms an ionic interaction with Lys85. interact with the backbone atoms of Asp133 and Val135 (Fig. 3A). The B ring of morin was protruding outward compared to ADP and The hydroxyl group at C-3 of the C ring overlaps with an oxygen forms a polar interaction. K. Kim et al. / Biochemical and Biophysical Research Communications 504 (2018) 519e524 523

Fig. 4. Surface plasmon resonance (SPR) of GSK3b and morin. The binding sensor grams of morin against GSK3b immobilized on the GLH sensor chip (left). The dissociation constant (Kd) values were calculated by an equilibrium analysis using the increasing concentration of the ligands (right).

We next compared the GSK3b/morin structure with the death- GSK3b was measured as ~67 mM, consistently indicating that associated protein kinase 1 (DAPK1)/morin structure, which is morin was not a strong inhibitor of GSK3b. However, if morin was another morin-kinase complex structure (PDB code: 5AUY). Inter- modified based on the structure presented in this study, the estingly, like GSK3b, DAPK1 also phosphorylated the tau protein derivatives would be stronger inhibitors. Thus, our structure and was associated in AD [21]. We assumed that morin has a syn- would provide a valuable starting point to design more potent ergetic role for AD, because it binds two AD-associating kinases, GSK3b inhibitors. GSK3b and DAPK1. DAPK1 is a positive mediator of g-interferon To date, many compounds that strongly inhibit GSK3b have induced programmed cell death, and the IC50 value when it is with been developed to control diverse human diseases, including AD. morin is 1.6 mM[22]. In the DAPK1/morin structure, morin is flipped The inhibitory compounds have stronger affinities than morin, by 180 around the A and C ring axes, and was deeper in the ATP and some of them have a complex structure with GSK3b [23]. binding pocket compared to morin in the GSK3b complex structure. However, it would be reasonable to control AD with functional The morin A and C rings show a similar binding pattern among foods rather than medicines because it is a slowly progressive GSK3b and DAPK1, but the hydroxyl group of C-40 in the morin B and non-fatal disease until its later stages. Morin can be regarded ring was definitely different. The C-4' of morin bound GSK3b binds as a safe ingredient from food and has been reported to have to the carboxyl group of Asn64, whereas those that bound DAPK1 many physiological effects [9,10]. Morin could show anti-AD ac- bound to the ε2 oxygen of Glu64 and the amino group of Phe162 tivity with minimal deleterious effects if it was taken every day (Fig. 3B). at a reasonable dose. Morin also has the ability to penetrate the bloodebrain barrier, which would make morin more promising as a functional food ingredient to prevent or delay AD [11]. 3.4. Binding affinity of morin to GSK3b Moreover, the mild inhibition of GSK3b would be beneficial for the control of type 2 diabetes mellitus because the inhibition of To measure the binding affinity of morin to GSK3b, we per- GSK3b is also involved in the activation of the insulin signaling formed SPR experiments. The N-terminally His6-tagged GSK3b [24]. protein was immobilized onto the NTA-coupled surface of the chip, and then 6 different concentrations of morin solution was passed over the chip. The kinetic results showed that the binding affinity Accession number was about 67 mM, indicating weak binding between morin and GSK3b (Fig. 4). This result was consistent with the previous result of The coordinates of GSK3b with morin and the structure factors a partial inhibition of GSK3b by treatment with 100 mM morin for have been deposited in the Protein Data Bank with the code as tau phosphorylation [11]. 6AE3.

4. Discussion Acknowledgements

This study presented the complex structure of GSK3b and We thank the staff scientists for assistance at the beamlines 5A morin at a 2.14 Å resolution, which provided the structural basis and 1A of the Photon Factory, and the beamline 5C, 7A, and 11C of for understanding the specificity of morin to GSK3b.Thehydro- Pohang Light Source. This work was supported by the National phobic adenine-binding site of GSK3b exhibited the ability to Research Foundation of Korea (NRF) grant funded by the Ministry of accommodate the molecular structure of the A-C ring of the Science and ICT (NRF-2016R1A2B2013305 to HSC, NRF- flavonoid. The ribose-binding pocket of GSK3b, linked with the 2016R1A5A1010764 to HSC, NRF-2017M3A9F6029755 to HSC and Gly-rich loop, also provides a platform for polar interaction with NCH) and by Korea Institute of Planning and Evaluation for Tech- a hydroxyl group in the B ring. However, since morin was rela- nology in Food, Agriculture, Forestry (IPET) through Agriculture, tively small, compared to the size of the nucleotide binding Food and Rural Affairs Research Center Support Program, funded by pocket, the number of the polar interactions involved in morin Ministry of Agriculture, Food and Rural Affairs (MAFRA) (710012- was less than that of ADP. The dissociation constant of morin to 03-1-HD120 to NCH). 524 K. Kim et al. / Biochemical and Biophysical Research Communications 504 (2018) 519e524

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