The latest version is at ...using pET-32b (+) vector and transformed into Rosetta host cells...

block interaction between S100A11 and RAGE V domain

1

Tranilast Blocks the Interaction between the Protein S100A11 and Receptor for Advanced

Glycation End Products (RAGE) V Domain and Inhibits Cell Proliferation

Yen-Kai Huang1, Ruey-Hwang Chou2 and Chin Yu1

From the 1Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan, 2Graduate

Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung

40454, Taiwan

Running title: block interaction between S100A11 and RAGE V domain

To whom correspondence should be addressed: Dept. of Chemistry, National Tsing Hua University, Hsinchu

30013, Taiwan. Fax: 886-3-5711082; Phone: 886-35-721524; E-mail: [email protected]

Keywords: S100 proteins, receptor for advanced glycation end products (RAGE), Tranilast, protein

purification, nuclear magnetic resonance (NMR), protein complex, cell proliferation

ABSTRACT

The human S100 calcium-binding protein A11

(S100A11) is a member of S100 protein family.

Once S100A11 proteins bind to calcium ions at EF-

hand motifs, S100A11 will change its

conformation promoting interaction with target

proteins. The receptor for advanced glycation end

products (RAGE) consists of three extracellular

domains, including V domain, C1 domain and C2

domain. In this case, V domain is the target for

mS100A11 binding. RAGE binds to the ligands

result in cell proliferation, cell growth and several

signal transduction cascades. We used nuclear

magnetic resonance (NMR) and fluorescence

spectroscopy to demonstrate the interactions

between mS100A11 and RAGE V domain. The

Tranilast molecule is a drug used for treating

allergic disorders. We found out that RAGE V

domain and Tranilast would interact with

mS100A11 by using 1H-15N HSQC NMR titrations.

According to the results, we obtained two binary

complex models from the HADDOCK program,

S100A11-RAGE V domain and S100A11-Tranilast,

respectively. We overlapped two binary complex

models with the same orientation of S100A11

homodimer and demonstrated that Tranilast would

block the binding site between S100A11 and

RAGE V domain. We further utilized the WST-1

assay to confirm this result. We think that the

results will be potentially useful in the

development of new anti-cancer drugs.

INTRODUCTION

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.722215The latest version is at JBC Papers in Press. Published on May 12, 2016 as Manuscript M116.722215

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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Human S100 proteins are a family of low-

molecular-weight, homodimeric and acidic

proteins which contain two EF-hand motifs that

can bind to calcium ions (1,2). Human S100A11

(S100C) protein is a member of S100 family.

S100A11 was first discovered in chicken gizzard

and is also called calgizzarin (3). S100A11 protein

can be expressed in heart, kidney, liver, lung and

other tissues. It is most abundant in smooth muscle

tissues (4). In previous studies, it has been shown

that S100A11 protein can interact with various

other proteins such as annexin A1 and A2, ATPase

Rad54B and RAGE. The interactions induce

activities or conformational changes of these target

proteins and further promote the specific

physiological functions. Calcium-bound S100A11

homodimer acts as a bridge to link two N-terminals

of annexin proteins inducing plasma membrane

vesiculation in cells (5,6). Interaction between

S100A11 and DNA repair and recombination

protein Rad54B participates in the repair of

double-strand DNA and regulation of cell cycle (7).

Furthermore, S100A11-RAGE interaction is

correlated to the inflammation of chondrocyte (8).

It has been reported that overexpression of

S100A11 protein is associated with a number of

cancers, including breast (9), colon (10), pancreatic

(11) and papillary thyroid carcinoma (12).

However, overexpression of S100A11 protein

result in the contrary effect in ovarian (13) and

bladder cancer (14). Reduced expression of

S100A11 protein is correlated with progression of

ovarian and bladder cancer which has been

investigated in the previous studies.

RAGE, the receptor of the immunoglobulin for

signal transductions at cell surface, is a 35 kDa

protein (15,16). RAGE consists of one cytoplasmic

domain at the C-terminus, one transmembrane

domain, two distinct constant domains (C1, C2),

and one variable domain at the N-terminus (V) (17).

The variable domain is predominantly related to

ligand binding such as seen in most S100 family

proteins (18), advanced glycation end products

(AGEs) (19,20), amyloid-β protein (21) and

amphoterin (HMGB1) (22,23). Some ligands can

interact with both the variable domain and constant

domain simultaneously, such as S100B (24) and

S100A12 protein (25,26). Furthermore, S100A6

protein can even interact with V, C1 and C2 domain

(27,28). However, the mechanism by which some

S100 family proteins bind to RAGE are still

shrouded in mystery. The cytoplasmic domain is a

tail domain with various charged residues which

are correlated to autophosphorylation and

activation of intracellular signal transduction

pathways (29). Once ligands bind to the RAGE, it

would trigger homodimerization of two RAGEs,

followed by mutual phosphorylation of two

cytoplasmic domains and signal transduction

cascades (30-32). These signal transductions

induce inflammation, cell proliferation and

migration and even tumor growth. However, which

pathway would be activated depend on the type and

concentration of binding ligands in different cells

(33). RAGE has been reported in many diseases

such as Alzheimer’s disease (34), diabetic

complication (35), chronic vascular inflammation

(36) and even some cancers (37-39). Therefore,

insight into the interactions between RAGE and its

ligands is important for development of treatments


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to RAGE-dependent diseases.

The Tranilast molecule (N-(3, 4-

dimethoxycinnamoyl) anthranilic acid) is a drug

used for anti-allergy treatment (40). Tranilast was

initially developed to treat children bronchial

asthma (41). Subsequently, it also has been used for

the treatment of other allergic disorders such as

allergic rhinitis (42) and allergic dermatitis (43).

Recently, Tranilast has been developed to be an

anti-proliferative drug for neurofibroma cells in

vitro (44).

Different S100 proteins were reported to bind to

different domains of RAGE. However, V domain

of RAGE was reported to bind different S100

proteins. For example, S100B were reported to

bind V domain (24,45). With SPR experiment,

Heizmann showed that S100A2, S100A5 and

S100A12 bound to V domain of RAGE (18).

Although the interaction between S100A11 and

RAGE has been mentioned in previous studies,

they are not at the molecular level (46). In this study,

we seek to find out insight into the interaction of

S100A11 with RAGE V domain and construct a

model for demonstrating a S100A11-RAGE V

domain heterotetrameric complex at the molecular

level. Binding interfaces of mS100A11 and RAGE

V domain are identified using NMR spectroscopy

(47). We determined the binding constant (Kd) of

the mS100A11-RAGE V domain complex by

fluorescence spectroscopy. The HADDOCK

program was used to approach the residues at the

interfaces of two proteins we obtained from NMR

1H-15N HSQC titrations (48). Furthermore,

Tranilast was identified to be an inhibitor that could

interact with mS100A11 and block the interface of

S100A11 to RAGE V domain in this study. These

were sustained by the results of NMR 1H-15N

HSQC titrations, fluorescence experiments and

HADDOCK modeling. We further proved the

inhibition properties of Tranilast by WST-1 assay

(49). The putative complex models and result of

WST-1 assay provide the prospective insight into

the new drug development of treatments for

RAGE-dependent diseases.

EXPERIMENTAL PROCEDURES

Mutant S100A11 (mS100A11) expression and

purification

There are two free cysteines in S100A11 molecule.

In this case, dithiothreitol (DTT) must be used in

the buffer for NMR experiments to ensure that

S100A11 contains cysteines in its free form.

However, when the structure determining of

S100A11-RAGE V domain complex is performed

by NMR, DTT should be avoided because RAGE

V domain contains one disulfide bond. This

disulfide bond would be broken by DTT. In order

to solve this problem, we constructed the mutant

S100A11 (mS100A11). Residue Cys13 and Cys91

were mutated to serine, so that the NMR

experiments could be executed without DTT. Since

the 1H-15N HSQC spectra of wild type and mutant

form of S100A11 are similar, we believe that the

three-dimensional structure of mutant and wild-

type are alike. The cDNA of the mutant S100A11

(C13S and C91S) was constructed and purchased

from Mission Biotech Company. We used pET20b

(+) as a vector which was transformed and

overexpressed in Escherichia coli BL21 host cells

(Novagen). We prepared 15N-labeled mS100A11

protein and cultured the E. coli cells in M9 medium,


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containing 15N-ammonium chloride as the sole

nitrogen source. The cells were cultured at 310 K

until the optical density (OD) reached about 0.7 at

600 nm, and then 1 mM isopropyl β-D-

thiogalactopyranoside (IPTG) was added for the

induction of protein expression with 200 rpm

shaking at 298 K. After 21 h of overnight induction,

the cells were then centrifuged at 6,000 rpm for 20

min, followed by resuspension in buffer A (25 mM

Tris-HCl, 100 mM NaCl, 5 mM CaCl2, pH 7.5).

The cell lysis was performed three times by using

the French press at 1,000 psi. We removed the

lysate by centrifugation at 13,000 rpm and 4 °C for

45 min. The supernatant was then purified by Hi-

Prep Phenyl FF 16/10 column on the AKTA FPLC

system (GE Healthcare). The unwanted proteins

were washed out by buffer A, and mS100A11

would bind to the column because of hydrophobic

interactions. After that, buffer B (elution buffer

containing 25 mM Tris-HCl, 100 mM NaCl, 5 mM

EDTA, pH 7.5) was used to detach the target

protein mS100A11 from the column. Finally, we

used a Hi-load 16/60 superdex 75 column (GE

Healthcare) that was equilibrated with buffer A as

the second column for buffer exchange and final

purification.

RAGE V domain expression and purification

Recombination of RAGE V domain was performed

using pET-32b (+) vector and transformed into

Rosetta host cells (Novagen). 15N-labeled RAGE V

domain proteins were also prepared in M9 medium.

The cells were cultured at 310 K until the OD

reached 0.7, then we added 1 mM IPTG for

induction with 200 rpm shaking at 298 K for 3 h.

After induction, the cells were centrifuged at 6,000

rpm for 20 min, followed by resuspension in buffer

C (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). The

cell lysis was performed by using the sonicator

(Qsonica) for 45 min in an ice bath. The lysate was

removed by centrifugation at 13,000 rpm and 4 °C

for 45 min. The supernatant was purified using a Ni

sepharoseTW 6 fast flow column (GE Healthcare)

which was designed for scaling up purification of

histidine-tagged proteins. At first, unwanted

impurities which had no binding affinity with the

Ni column were washed out with 50 mL buffer C.

Then we used 50 mL buffer D (20 mM Tris-HCl,

300 mM NaCl, 20 mM imidazole, pH 8.0) to wash

out the unwanted proteins which had weak binding

with Ni column, followed by elution of the target

protein, which shows strong binding with Ni

column using 50 mL buffer E (elution buffer 20

mM Tris-HCl, 300 mM NaCl, 500 mM imidazole,

pH 8.0). We concentrated the collection of elution

to 2 mL, and then the His-tag which contained six

continuous histidine units at the N-terminus, was

removed by using thrombin cleavage (1 unit per mg

of protein) in a water bath at 25 °C for 3 h. After

that, the RAGE V domain-containing fraction was

purified using HITACHI L-7000 system HPLC

with Atlantis dc18 Prep Column (Waters) as the

final purification. Both molecular weights of

mS100A11 and RAGE V domain proteins were

confirmed by ESI-MASS spectrometry.

NMR 1H-15N HSQC titration experiments

All NMR HSQC data were acquired using the

Varian 700 MHz NMR spectrometer at 298 K. All

titrations were performed under the same buffer

condition (25 mM Tris-HCl, 100 mM NaCl, 5 mM

CaCl2, pH 7.5, 10 % D2O). Unlabeled RAGE V


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domain was added to uniformly 15N-labeled

mS100A11 at molar ratios of 0:1, 0.25:1, 0.5:1,

0.75:1 and 1:1. The reverse titration experiments

(15N-labeled RAGE V domain adding unlabeled

mS100A11) were performed at 0:1, 0.25:1, 0.5:1,

0.75:1, 1:1, 1.5:1 and 2:1. On the other hand, the

titration of Tranilast and 15N-labeled mS100A11

was performed at molar ratios of 0:1, 0.5:1, 1:1, 2:1

and 3:1. After overlapping series of the HSQC

titration spectrums, we could calculate intensity

reductions or chemical shift perturbations of cross-

peaks by plotting bar diagrams.

Fluorescence experiments

All the fluorescence experiments were performed

under the same buffer conditions; using buffer A.

RAGE V domain has three tryptophan residues

which could be excited and emit fluorescence. The

fluorescence experiments were performed using

the F-2500 fluorescence spectrophotometer

(HITAICHI). We used wavelength of 295 nm as the

excitation light source, and RAGE V domain has a

maximum emission band at a wavelength of 351

nm. We detected the wavelength of emission in the

range of 310 to 440 nm. The increasing

concentration of mS100A11 (0 μM, 0.085 μM, 0.17

μM, 0.255 μM, 0.34 μM, 0.425 μM, 0.51 μM, 0.595

μM, 0.68 μM, 0.765 μM, 0.85 μM, 0.935 μM, 1.02

μM, 1.105 μM, 1.190 μM, 1.275 μM, 1.36 μM,

1.445 μM, 1.530 μM, 1.615 μM,, 1.700 μM, 1.785

μM, 1.87 μM, 1.955 μM and 2.04 μM) were added

to RAGE V domain, which was at a concentration

of 0.68 μM.

Tranilast also has fluorescence emission. We found

that Tranilast has an absorption band at 310 nm.

Therefore, we used wavelength of 310 nm to be the

excitation light source, and Tranilast has a

maximum emission band at a wavelength of 335

nm. We added increasing concentration of

mS100A11 (0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM,

2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5

μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM,

9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM and 11.5

μM) to the Tranilast solution at a concentration of

5 μM.

The data we acquired from both fluorescence

experiments were plotted as 1/[ mS100A11] versus

1/(I − I0). We utilized the following equation to a fit

linear curve in the Origin program which was used

to calculate the dissociation constant of two sample

(50): 1

(I − I0)=

1

(I1 − I0)+

Kd(I1 − I0)

×1

[mS100A11]

or ΔF =ΔF𝑚𝑎𝑥×[𝑚𝑆100𝐴11]

𝐾𝑑+[𝑚𝑆100𝐴11]

I0, I and I1 are representations of emission

intensities in the absence of mS100A11, in the

middle concentration of mS100A11 and in the

maximal concentration of mS100A11 we

performed, respectively. ΔF is the difference of

fluorescence intensity between I and I0. ΔFmax is the

maximal fluorescence change, and Kd stands for

the dissociation constant.

RESULT

Tryptophan residue fluorescence measurements

The intrinsic fluorescence of tryptophan residues

are very sensitive to structural changes of proteins

and the environmental polarity around the

tryptophan when substrates were binding (51).

Tryptophan residues exhibit maximum emission


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ranging from 340 to 360 nm with excitation at 295

nm in highly polar environments. However,

maximum emission of tryptophan residues is 330

to 350 nm in hydrophobic environments (52).

There are three tryptophan residues in RAGE V

domain, these being W51, W61 and W72

respectively. Excited by wavelength of 295 nm,

the maximum emission of RAGE V domain is at

351 nm (53). Residue W61 of RAGE V domain is

at the binding site, therefore we monitored the

change of fluorescence intensity at 351 nm upon

excitation at 295 nm. Increasing mS100A11

resulted in a decreasing RAGE V domain

fluorescence intensity in titration experiments

(FIGURE 1A). According to the curve we fitted

with the fluorescence intensity changes versus the

mS100A11 concentrations as one binding site

model, we obtained a dissociation constant (Kd) of

approximately 0.5 µM (FIGURE 1B). Overall, the

fluorescence results showed the binding between

RAGE V domain and mS100A11 that is in the

lower micromolar level, providing that the

conformation of the protein complex is very stable

under normal conditions of physiology.

Tranilast fluorescence measurements

Tranilast has two benzyl groups, so that it also has

fluorescence emission. We monitored the change of

Tranilast fluorescence intensity at 335 nm upon

excitation at 310 nm. The increasing fluorescence

intensities of Tranilast were detected by increasing

the concentration of mS100A11 (FIGURE 1C).

The dissociation constant we obtained was 11.75

µM (FIGURE 1D). This dissociation constant is

also present at the micromolar level, indicating the

formation of a stable complex.

Molecular docking

We used the high ambiguity driven protein-protein

docking (HADDOCK) program to dock S100A11

and RAGE V domain (or Tranilast) and obtain

binary S100A11-RAGE V domain (or S100A11-

Tranilast) complex model (54). In this case, we

utilized the structure of wild-type S100A11 (PDB

ID: 2LUC) taken from the Protein Data Bank as the

structure of mS100A11 (55). The 3D structure of

RAGE V domain was also taken from the Protein

Data Bank (PDB ID: 2E5E) (56). The three-

dimensional structure of Tranilast was taken from

DrugBank (Accession Number: DB07615), and its

structure was given for reference (FIGURE 2). The

signals of residues that exhibited chemical shift

perturbations or intensities decreasing on HSQC

titration experiments were performed. It indicated

that these residues were located in the interface

region of two proteins. We used HADDOCK to

identify these residues as ambiguous interaction

restrain. The active and passive residues of the

proteins were predicted by the NACCESS program

(57). NACCESS defines that the area of residues

for accessible surface greater than 30 %, including

sidechain and backbone, are active residues. In

contrast, the area less than 30 % are defined as the

passive residues. For docking S100A11 with

RAGE V domain, the 2,000 total structures were

set for rigid body docking by standard HADDOCK

protocol with optimized potential for liquid

simulation (OPLSX) parameters (58). Then 800

structures were set for semi-flexible refinement,

and the 200 lowest-energy structures were

analyzed. On the other hand, the 8,000 total

structures for rigid-body docking, 1,000 structures


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for semi-flexible refinement and the 800 lowest-

energy structures were finally analyzed for docking

S100A11 with Tranilast. Results of the calculation

would be classified into clusters which contained

similar structures with small root mean square

distance (RMSD). We demonstrated the results of

structures on the PyMOL program (59).

mS100A11 and RAGE V domain binding

interface

Using the two-dimensional 1H-15N HSQC NMR

spectrum is very useful for identifying the binding

interface of the protein-protein complex (60). In the

interfacial residues of Ca2+-mS100A11 and RAGE

V domain complex, they were observed that the

chemical shift perturbations and intensities of

peaks were changed compared to free mS100A11

without RAGE V domain on 1H-15N HSQC. We

overlapped HSQC of 15N-labeled free mS100A11

and of the 15N-labeled mS100A11 complex with

unlabeled RAGE V domain (FIGURE 3A).

Intensities decreasing and chemical shift

perturbations of NMR signals were observed at the

ratio of 1:1 for 15N-mS100A11 complex with

unlabeled RAGE V domain. The decreasing and

shift perturbations of signals result from complex

formation, which makes the environment of the

residues change at the binding interface. This

formation caused an obvious decrease in the

intensities and perturbations of amide cross peaks

on 1H-15N HSQC spectrums. In order to find out

which residues interacted with RAGE V domain,

the peak intensities of 15N-labeled mS100A11 in

complex with unlabeled RAGE V domain (I) on

1H-15N HSQC compared to free mS100A11 (I0) and

chemical shift perturbations were plotted in bar

diagrams (FIGURE 3B and 3C respectively). The

equation for the perturbation calculation was

derived by the NMR spectroscopy research group

of Utrecht University (61). Bar diagram analyses

indicated that most of residues which exhibited

decreasing intensities or perturbations located at

the hydrophobic region of mS100A11, including

A48, A49, T51, K52, N53, Q54, D68, E79, F80,

G85, L87, A88 and A90. These residues are

colored in red (FIGURE 3D). Most of these

residues are at the helix-2 and 4 of S100A11. In

reverse titration, we observed signal decreasing on

1H-15N HSQC only using 15N-labeled RAGE V

domain with unlabeled mS100A11 at the ratio of

1:1 (FIGURE 4A). We also used a bar diagram to

find the affected residues such as T55, G56, W61,

K62, V63, L64, S65, D73, G95 and R98 (FIGURE

4B). These residues were labeled in red on the

structure of RAGE V domain (FIGURE 4C). The

role of these residues which are accessible for

solvent predicted by NACCESS was further

substantiated by the analysis of S100A11 complex

with RAGE V domain. Overall, labeled residues on

each protein exhibited the binding interface of

protein-protein complex.

The S100A11-RAGE V domain complex model

By HSQC titration experiments, we identified the

binding regions of proteins and looked for the

structural model of the S100A11 in complex with

RAGE V domain. Most these residues of

mS100A11 are buried in the hydrophobic site

forming a binding interface upon calcium binding.

Most affected residues are also form a continuous

binding interface in the structure of RAGE V

domain. We set these two binding interfaces of


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proteins to form a binary complex model using

HADDOCK 2.2. The 2,000 complex structures

were produced from using rigid-body minimization,

followed by further water refinement to propose

the best 200 structures with lowest total energy. We

chose 20 backbone structures with the lowest-

energy were overlapped to demonstrate the

similarity of the calculated structures (FIGURE

5A). The residues of mS100A11, N53, Q54 and

A90 (painted in red) interact with residues W61,

V63, S65 and D73 of RAGE V domain (painted in

yellow) are shown in FIGURE 5B. We used the

PROCHECK analysis of European Bioinformatics

Institute (EBI) on their website to check the

structure stereochemistry of complex (62).

Ramachandran plot and the results show that area

of the disallowed regions is only 0.6 % and that of

the most favoured regions is about 81.1 %. The

overall score average of G-Factors is 0.13; it is thus

in the usual range (FIGURE S4A and Table S1).

Binding interface between mS100A11 and

Tranilast

The residues at the interface of Ca2+-mS100A11 in

complex with Tranilast were observed that only

chemical shifts were changed compared to free

mS100A11 without Tranilast in 1H-15N HSQC

spectra. We overlapped HSQC of 15N-labeled free

mS100A11 and 15N-labeled mS100A11 complex

with Tranilast (FIGURE 6A). In this case, no

decreasing signals were observed and thus we can

choose the perturbed residues directly. We found

that most residues were located at the helix-2,

helix-3 and helix-4 of S100A11. These residues are

colored in red for L47, A48, T51, N53, L60, R62,

L87 and A90 (FIGURE 6B).

Structural model of the mS100A11 in complex

with Tranilast molecular

Most affected residues of mS100A11 are also at the

hydrophobic site and form a continuous interface

to interact with Tranilast. We set S100A11 and

Tranilast molecular to form binary complex model

using the HADDOCK with restrains obtained from

chemical shift perturbations in HSQC spectrums.

The 8,000 complex structures were produced from

rigid-body minimization, followed by further water

refinement to the best 800 structures with the

lowest total energy. HADDOCK analyzed these

800 lowest-energy structures and group them into a

number of clusters. We chose the complex with the

best symmetry between two Tranilast molecules in

the first cluster and demonstrated S100A11 in

complex with Tranilast on the PyMOL. Labeling

affected residues on the S100A11 homodimer

reveals that two Tranilast molecular bind to the

hydrophobic site of each S100A11 monomer

symmetrically (FIGURE 7A). The residues of

mS100A11, N53, L87 and A90 interact with

Tranilast (FIGURE 7B). We also used the

PROCHECK analysis to check the structure

stereochemistry of S100A11 in complex with

Tranilast. We produced a Ramachandran plot and

the results show that the area of the disallowed

regions is 0.0 % and that of the most favoured

regions is about 81.9 %. The overall score average

of G-Factors is - 0.04; it is also in the usual range

(FIGURE S4B and Table S2).

Water-soluble tetrazolium-1 (WST-1) cell

proliferation assay

For cell proliferation assay, WST-1 (2-(4-

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disulfophenyl)-2H-tetrazolium, monosodium salt)

was used as a reagent for the experiments. The

SW480 cell is a cell line with high expression of

RAGE and low expression of S100A11 protein.

Therefore, we can clearly observe cell activity

change and prevent the influence of endogenous

S100A11 in this cell. The increasing cell activity

was observed with increasing concentration of

exogenous mS100A11 (FIGURE 8, LANE 2-4).

However, the cell activity was reduced upon adding

exogenous RAGE V domain (FIGURE 8, LANE 5).

The reducing resulted from the competition of

exogenous RAGE V domain with endogenous

RAGE V domain. This observation can prove that

the signal transduction pathways indeed conduct by

the interaction between S100A11 and RAGE V

domain. On the other hand, we added 1 μM

Tranilast to 100 nM mS100A11 and compared this

to free 100 nM mS100A11. We observed

decreasing cell activity in mS100A11 with

Tranilast compared to free mS100A11 (FIGURE 8,

LANE 6). This indicated that Tranilast can

successfully act as an inhibitor of cell proliferation

for mS100A11-endogenous RAGE V domain

interaction.

DISCCUSIONS

The calcium-binding S100A11 contains two EF-

hand motifs which are located at the helix-1-loop-

helix-2 and helix-3-loop-helix-4 regions. These

regions mediate many signal transduction cascades

which are calcium-dependent. Thus, S100A11 is

associated with a large number of functions,

including intracellular and extracellular functions

in various cell types (63). The binding of calcium

ion to S100A11 homodimeric protein results in

conformational changes that promote the

interaction between the hydrophobic site of

S100A11 and target proteins and further mediate

activity of proteins (64). S100 protein family is one

of ligands of RAGE. The binding of one site of

S100A11 dimer to RAGE V domain induces

homodimerization of two adjacent RAGEs and

autophosphorylation of their cytoplasmic domains.

This autophosphorylation would trigger a series of

signal transduction cascades, leading to cell

proliferation and survival (FIGURE 9). The

interactions between mS100A11 and RAGE V

domain were characterized by using techniques

such as fluorescence spectroscopy and NMR

spectroscopy. The NMR titration experiments and

plotting bar diagrams identified the residues of

binding sites of mS100A11 homodimer and RAGE

V domain. Mapping these affected residues on

three-dimensional structures elucidates the binding

sites between mS100A11 and RAGE V domain.

According to the experimental results, we utilized

the HADDOCK program to calculate the

S100A11-RAGE V domain heterotetrameric

complex model which demonstrated the binding

sites between mS100A11 and RAGE V domain.

The results shows that the binding site of S100A11

is at helix-2 and helix-4. This region is similar to

the regions that have been reported as the binding

interfaces of other S100 family proteins complex

with RAGE V domain. For instance, S100P

homodimer was shown to interact with RAGE V

domain by helix-1 of one of monomer and helix-4’

of another monomer in previous study (65). It has

been reported that the binding site of S100A6 to

RAGE V domain is located at loop-1 and 3 and


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helix-4 (27). For S100B, the binding region on

loop-1, loop-3, helix-3 and helix-4 has been

investigated to determine its’ interaction with

RAGE V domain (24). These S100 proteins

generally have similar regions in binding interfaces

as a result of their high structural similarity in three

dimensions. Some parts of minor differences in

binding site between each S100 protein and RAGE

V domain may result from distinct identification of

RAGE V domain to S100 proteins due to the

differences in hydrophobic region, polarity, net

charge or properties of S100 proteins. Overall, our

results reveal that S100A11 homodimer interacts

with two RAGE V domain molecules

symmetrically and form a heterotetrameric

complex model. This model of S100A11-RAGE V

domain complex would be useful for designing

drugs to block the interaction between mS100A11

and RAGE V domain. Furthermore, we found out

that the Tranilast molecular could be a potential

drug for blocking the interaction between

mS100A11 and RAGE V domain based on our

results of experiments, including fluorescence,

NMR, HADDOCK modeling and WST-1 cell

proliferation assay. Figure 10 showed the

overlapped of following two complexes that

generated by our HADDOCK results: (1)

S100A11-RAGE V domain complex, painted in

green, and (2) S100A11-Tranilast complex

(S100A11 is painted in green and Tranilast

molecules are painted in multi-color). It clearly

showed that Tranilast blocked the binding sites

between mS100A11 and RAGE V domain. Our

findings give prominence to the inhibition of cell

proliferation resulting from the blocking

interaction of S100A11 with RAGE using Tranilast

molecule. This will also be helpful for improving

therapeutic strategies to treat RAGE-dependent

diseases or even cancers.

Acknowledgments: We thank members of the 700 MHz Nuclear Magnetic Resonance facility,

Instrumentation Center, Chemistry Department, National Tsing Hua University, Hsinchu, Taiwan and ESI-

MS, Instrumentation Center, Chemistry Department, National Chiao Tung University, Hsinchu, Taiwan.

The atomic coordinates and structure factors (codes 2LUC and 2E5E) have been deposited in the Protein

Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ

(http://www.rcsb.org/).

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this

article.

Author contributions: YKH, RHC and CY conceived and designed the study. YKH purified mS100A11

and RAGE V domain proteins and obtained complex models by using HADDOCK. RHC conducted WST-

1 assay experiments. YKH and RHC acquired and analyzed the data of results. YKH wrote the paper. All


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authors reviewed the results and approved the final version of the manuscript.

REFERENCES

1. Schafer, B. W., and Heizmann, C. W. (1996) The S100 family of EF-hand calcium-binding

proteins: functions and pathology. Trends Biochem Sci 21, 134-140

2. Heizmann, C. W., Fritz, G., and Schafer, B. W. (2002) S100 proteins: structure, functions

and pathology. Front Biosci 7, d1356-1368

3. Allen, B. G., Durussel, I., Walsh, M. P., and Cox, J. A. (1996) Characterization of the Ca2+-

binding properties of calgizzarin (S100C) isolated from chicken gizzard smooth muscle.

Biochem Cell Biol 74, 687-694

4. Schonekess, B. O., and Walsh, M. P. (1997) Molecular cloning and expression of avian

smooth muscle S100A11 (calgizzarin, S100C). Biochem Cell Biol 75, 771-775

5. Dempsey, A. C., Walsh, M. P., and Shaw, G. S. (2003) Unmasking the annexin I interaction

from the structure of Apo-S100A11. Structure 11, 887-897

6. Rintala-Dempsey, A. C., Santamaria-Kisiel, L., Liao, Y., Lajoie, G., and Shaw, G. S. (2006)

Insights into S100 target specificity examined by a new interaction between S100A11 and

annexin A2. Biochemistry 45, 14695-14705

7. Murzik, U., Hemmerich, P., Weidtkamp-Peters, S., Ulbricht, T., Bussen, W., Hentschel, J.,

von Eggeling, F., and Melle, C. (2008) Rad54B targeting to DNA double-strand break

repair sites requires complex formation with S100A11. Mol Biol Cell 19, 2926-2935

8. Cecil, D. L., Johnson, K., Rediske, J., Lotz, M., Schmidt, A. M., and Terkeltaub, R. (2005)

Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced

glycation end products. J Immunol 175, 8296-8302

9. Liu, X. G., Wang, X. P., Li, W. F., Yang, S., Zhou, X., Li, S. J., Li, X. J., Hao, D. Y., and

Fan, Z. M. (2010) Ca2+-binding protein S100A11: a novel diagnostic marker for breast

carcinoma. Oncol Rep 23, 1301-1308

10. Meding, S., Balluff, B., Elsner, M., Schone, C., Rauser, S., Nitsche, U., Maak, M., Schafer,

A., Hauck, S. M., Ueffing, M., Langer, R., Hofler, H., Friess, H., Rosenberg, R., and Walch,

A. (2012) Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel

markers for regional lymph node metastasis in colon cancer. J Pathol 228, 459-470

11. Xiao, M. B., Jiang, F., Ni, W. K., Chen, B. Y., Lu, C. H., Li, X. Y., and Ni, R. Z. (2012)

High expression of S100A11 in pancreatic adenocarcinoma is an unfavorable prognostic

marker. Med Oncol 29, 1886-1891

12. Anania, M. C., Miranda, C., Vizioli, M. G., Mazzoni, M., Cleris, L., Pagliardini, S.,

Manenti, G., Borrello, M. G., Pierotti, M. A., and Greco, A. (2013) S100A11

overexpression contributes to the malignant phenotype of papillary thyroid carcinoma. J


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


12

Clin Endocrinol Metab 98, E1591-1600

13. Liu, Y., Han, X., and Gao, B. (2015) Knockdown of S100A11 expression suppresses

ovarian cancer cell growth and invasion. Exp Ther Med 9, 1460-1464

14. Memon, A. A., Sorensen, B. S., Meldgaard, P., Fokdal, L., Thykjaer, T., and Nexo, E. (2005)

Down-regulation of S100C is associated with bladder cancer progression and poor survival.

Clin Cancer Res 11, 606-611

15. Schmidt, A. M., Vianna, M., Gerlach, M., Brett, J., Ryan, J., Kao, J., Esposito, C., Hegarty,

H., Hurley, W., Clauss, M., and et al. (1992) Isolation and characterization of two binding

proteins for advanced glycosylation end products from bovine lung which are present on

the endothelial cell surface. J Biol Chem 267, 14987-14997

16. Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F., Pan, Y. C., Elliston, K., Stern,

D., and Shaw, A. (1992) Cloning and expression of a cell surface receptor for advanced

glycosylation end products of proteins. J Biol Chem 267, 14998-15004

17. Dattilo, B. M., Fritz, G., Leclerc, E., Kooi, C. W., Heizmann, C. W., and Chazin, W. J.

(2007) The extracellular region of the receptor for advanced glycation end products is

composed of two independent structural units. Biochemistry 46, 6957-6970

18. Leclerc, E., Fritz, G., Vetter, S. W., and Heizmann, C. W. (2009) Binding of S100 proteins

to RAGE: an update. Biochim Biophys Acta 1793, 993-1007

19. Nakamura, K., Yamagishi, S., Nakamura, Y., Takenaka, K., Matsui, T., Jinnouchi, Y., and

Imaizumi, T. (2005) Telmisartan inhibits expression of a receptor for advanced glycation

end products (RAGE) in angiotensin-II-exposed endothelial cells and decreases serum

levels of soluble RAGE in patients with essential hypertension. Microvasc Res 70, 137-

141

20. Ott, C., Jacobs, K., Haucke, E., Navarrete Santos, A., Grune, T., and Simm, A. (2014) Role

of advanced glycation end products in cellular signaling. Redox Biol 2, 411-429

21. Deane, R., Du Yan, S., Submamaryan, R. K., LaRue, B., Jovanovic, S., Hogg, E., Welch,

D., Manness, L., Lin, C., Yu, J., Zhu, H., Ghiso, J., Frangione, B., Stern, A., Schmidt, A.

M., Armstrong, D. L., Arnold, B., Liliensiek, B., Nawroth, P., Hofman, F., Kindy, M., Stern,

D., and Zlokovic, B. (2003) RAGE mediates amyloid-beta peptide transport across the

blood-brain barrier and accumulation in brain. Nat Med 9, 907-913

22. Hori, O., Brett, J., Slattery, T., Cao, R., Zhang, J., Chen, J. X., Nagashima, M., Lundh, E.

R., Vijay, S., Nitecki, D., and et al. (1995) The receptor for advanced glycation end products

(RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-

expression of rage and amphoterin in the developing nervous system. J Biol Chem 270,

25752-25761

23. Sims, G. P., Rowe, D. C., Rietdijk, S. T., Herbst, R., and Coyle, A. J. (2010) HMGB1 and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


13

RAGE in inflammation and cancer. Annu Rev Immunol 28, 367-388

24. Koch, M., Chitayat, S., Dattilo, B. M., Schiefner, A., Diez, J., Chazin, W. J., and Fritz, G.

(2010) Structural basis for ligand recognition and activation of RAGE. Structure 18, 1342-

1352

25. Srikrishna, G., Nayak, J., Weigle, B., Temme, A., Foell, D., Hazelwood, L., Olsson, A.,

Volkmann, N., Hanein, D., and Freeze, H. H. (2010) Carboxylated N-glycans on RAGE

promote S100A12 binding and signaling. J Cell Biochem 110, 645-659

26. Xie, J., Burz, D. S., He, W., Bronstein, I. B., Lednev, I., and Shekhtman, A. (2007)

Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end

products (RAGE) using symmetric hydrophobic target-binding patches. J Biol Chem 282,

4218-4231

27. Mohan, S. K., Gupta, A. A., and Yu, C. (2013) Interaction of the S100A6 mutant (C3S)

with the V domain of the receptor for advanced glycation end products (RAGE). Biochem

Biophys Res Commun 434, 328-333

28. Leclerc, E., Fritz, G., Weibel, M., Heizmann, C. W., and Galichet, A. (2007) S100B and

S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor

for advanced glycation end products) immunoglobulin domains. J Biol Chem 282, 31317-

31331

29. Hudson, B. I., Kalea, A. Z., Del Mar Arriero, M., Harja, E., Boulanger, E., D'Agati, V., and

Schmidt, A. M. (2008) Interaction of the RAGE cytoplasmic domain with diaphanous-1 is

required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42. J

Biol Chem 283, 34457-34468

30. Rouhiainen, A., Kuja-Panula, J., Tumova, S., and Rauvala, H. (2013) RAGE-mediated cell

signaling. Methods Mol Biol 963, 239-263

31. Kierdorf, K., and Fritz, G. (2013) RAGE regulation and signaling in inflammation and

beyond. J Leukoc Biol 94, 55-68

32. Saleh, A., Smith, D. R., Tessler, L., Mateo, A. R., Martens, C., Schartner, E., Van der Ploeg,

R., Toth, C., Zochodne, D. W., and Fernyhough, P. (2013) Receptor for advanced glycation

end-products (RAGE) activates divergent signaling pathways to augment neurite

outgrowth of adult sensory neurons. Exp Neurol 249, 149-159

33. Donato, R. (2007) RAGE: a single receptor for several ligands and different cellular

responses: the case of certain S100 proteins. Curr Mol Med 7, 711-724

34. Yan, S. D., Chen, X., Fu, J., Chen, M., Zhu, H., Roher, A., Slattery, T., Zhao, L., Nagashima,

M., Morser, J., Migheli, A., Nawroth, P., Stern, D., and Schmidt, A. M. (1996) RAGE and

amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382, 685-691

35. Stern, D. M., Yan, S. D., Yan, S. F., and Schmidt, A. M. (2002) Receptor for advanced


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


14

glycation endproducts (RAGE) and the complications of diabetes. Ageing Res Rev 1, 1-15

36. Basta, G., Schmidt, A. M., and De Caterina, R. (2004) Advanced glycation end products

and vascular inflammation: implications for accelerated atherosclerosis in diabetes.

Cardiovasc Res 63, 582-592

37. Logsdon, C. D., Fuentes, M. K., Huang, E. H., and Arumugam, T. (2007) RAGE and

RAGE ligands in cancer. Curr Mol Med 7, 777-789

38. Kuniyasu, H., Oue, N., Wakikawa, A., Shigeishi, H., Matsutani, N., Kuraoka, K., Ito, R.,

Yokozaki, H., and Yasui, W. (2002) Expression of receptors for advanced glycation end-

products (RAGE) is closely associated with the invasive and metastatic activity of gastric

cancer. J Pathol 196, 163-170

39. Sparvero, L. J., Asafu-Adjei, D., Kang, R., Tang, D., Amin, N., Im, J., Rutledge, R., Lin,

B., Amoscato, A. A., Zeh, H. J., and Lotze, M. T. (2009) RAGE (Receptor for Advanced

Glycation Endproducts), RAGE ligands, and their role in cancer and inflammation. J Transl

Med 7, 17

40. Komatsu, H., Kojima, M., Tsutsumi, N., Hamano, S., Kusama, H., Ujiie, A., Ikeda, S., and

Nakazawa, M. (1988) Study of the mechanism of inhibitory action of tranilast on chemical

mediator release. Jpn J Pharmacol 46, 43-51

41. Shioda, H. (1979) A double blind controlled trial of N-(3',4'-dimethoxycinnamoyl)

anthranilic acid on children with bronchial asthma. N-5' Study Group in Children. Allergy

34, 213-219

42. Okuda, M., Ishikawa, T., Saito, Y., Shimizu, T., and Baba, S. (1984) A clinical evaluation

of N-5' with perennial-type allergic rhinitis--a test by the multi-clinic, intergroup, double-

blind comparative method. Ann Allergy 53, 178-185

43. Kondo, N., Fukutomi, O., Kameyama, T., and Orii, T. (1992) Inhibition of proliferative

responses of lymphocytes to food antigens by an anti-allergic drug, N(3',4'-

dimethoxycinnamoyl) anthranilic acid (Tranilast) in children with atopic dermatitis. Clin

Exp Allergy 22, 447-453

44. Yamamoto, M., Yamauchi, T., Okano, K., Takahashi, M., Watabe, S., and Yamamoto, Y.

(2009) Tranilast, an anti-allergic drug, down-regulates the growth of cultured neurofibroma

cells derived from neurofibromatosis type 1. Tohoku J Exp Med 217, 193-201

45. Ostendorp, T., Leclerc, E., Galichet, A., Koch, M., Demling, N., Weigle, B., Heizmann, C.

W., Kroneck, P. M., and Fritz, G. (2007) Structural and functional insights into RAGE

activation by multimeric S100B. Embo j 26, 3868-3878

46. Sakaguchi, M., Sonegawa, H., Murata, H., Kitazoe, M., Futami, J., Kataoka, K., Yamada,

H., and Huh, N. H. (2008) S100A11, an dual mediator for growth regulation of human

keratinocytes. Mol Biol Cell 19, 78-85


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


15

47. Walters, K. J., Gassner, G. T., Lippard, S. J., and Wagner, G. (1999) Structure of the soluble

methane monooxygenase regulatory protein B. Proc Natl Acad Sci U S A 96, 7877-7882

48. de Vries, S. J., van Dijk, M., and Bonvin, A. M. (2010) The HADDOCK web server for

data-driven biomolecular docking. Nat Protoc 5, 883-897

49. Berridge, M. V., Herst, P. M., and Tan, A. S. (2005) Tetrazolium dyes as tools in cell biology:

new insights into their cellular reduction. Biotechnol Annu Rev 11, 127-152

50. Fernandez-Fernandez, M. R., Veprintsev, D. B., and Fersht, A. R. (2005) Proteins of the

S100 family regulate the oligomerization of p53 tumor suppressor. Proc Natl Acad Sci U S

A 102, 4735-4740

51. Lakowicz, J. R. (2013) Principles of fluorescence spectroscopy, Springer Science &

Business Media

52. Vivian, J. T., and Callis, P. R. (2001) Mechanisms of tryptophan fluorescence shifts in

proteins. Biophys J 80, 2093-2109

53. Gospodarska, E., Kupniewska-Kozak, A., Goch, G., and Dadlez, M. (2011) Binding studies

of truncated variants of the Abeta peptide to the V-domain of the RAGE receptor reveal

Abeta residues responsible for binding. Biochim Biophys Acta 1814, 592-609

54. Dominguez, C., Boelens, R., and Bonvin, A. M. (2003) HADDOCK: a protein-protein

docking approach based on biochemical or biophysical information. J Am Chem Soc 125,

1731-1737

55. Hung, K. W., Chang, Y. M., and Yu, C. (2012) NMR structure note: the structure of human

calcium-bound S100A11. J Biomol NMR 54, 211-215

56. Matsumoto, S., Yoshida, T., Murata, H., Harada, S., Fujita, N., Nakamura, S., Yamamoto,

Y., Watanabe, T., Yonekura, H., Yamamoto, H., Ohkubo, T., and Kobayashi, Y. (2008)

Solution structure of the variable-type domain of the receptor for advanced glycation end

products: new insight into AGE-RAGE interaction. Biochemistry 47, 12299-12311

57. Murakami, Y., and Jones, S. (2006) SHARP2: protein-protein interaction predictions using

patch analysis. Bioinformatics 22, 1794-1795

58. Linge, J. P., Williams, M. A., Spronk, C. A., Bonvin, A. M., and Nilges, M. (2003)

Refinement of protein structures in explicit solvent. Proteins 50, 496-506

59. DeLano, W. L. (2002) The PyMOL molecular graphics system.

60. Williamson, M. P. (2013) Using chemical shift perturbation to characterise ligand binding.

Prog Nucl Magn Reson Spectrosc 73, 1-16

61. Dominguez, C., Bonvin, A. M., Winkler, G. S., van Schaik, F. M., Timmers, H. T., and

Boelens, R. (2004) Structural model of the UbcH5B/CNOT4 complex revealed by

combining NMR, mutagenesis, and docking approaches. Structure 12, 633-644

62. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


16

(1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein

structures solved by NMR. J Biomol NMR 8, 477-486

63. Donato, R., Cannon, B. R., Sorci, G., Riuzzi, F., Hsu, K., Weber, D. J., and Geczy, C. L.

(2013) Functions of S100 proteins. Curr Mol Med 13, 24-57

64. Santamaria-Kisiel, L., Rintala-Dempsey, A. C., and Shaw, G. S. (2006) Calcium-dependent

and -independent interactions of the S100 protein family. Biochem J 396, 201-214

65. Penumutchu, S. R., Chou, R. H., and Yu, C. (2014) Structural insights into calcium-bound

S100P and the V domain of the RAGE complex. PLoS One 9, e103947

FOOTNOTES

This work was supported by the Ministry of Science and Technology (MOST) Taiwan (Grant number

MOST 103-2113-M-007-017-MY3 to Chin Yu and 104-2320-B-039-031 to Ruey-Hwang Chou) and China

Medical University Hospital, Taiwan (Grant number CMU104-S-02 to Ruey-Hwang Chou).

The abbreviations used are: RAGE, the receptor for advanced glycation end products; HSQC, heteronuclear

single quantum correlation; HADDOCK, high ambiguity driven docking; HMGB1, high-mobility group

protein 1; DTT, dithiothreitol; E. coli, Escherichia coli; OD, optical density; IPTG, isopropyl β-D-

thiogalactopyranoside; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium dodecyl sulfate

polyacrylamide gel electrophoresis.


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FIGURE LEGENDS

FIGURE 1. Identify the binding affinities of mS100A11-RAGE V domain complex and mS100A11-

Tranilast complex by using fluorescence spectroscopy. A, The emission spectrums of 0.68 μM RAGE V

domain fluorescence titrations exhibit the decreasing fluorescence intensities with increasing concentration

of mS100A11 in micromolar level. B, The curve with fluorescence intensity changes versus mS100A11

concentrations by measuring at a wavelength of 351 nm. Dissociation constant (kd) is 0.5 μM calculated

from the equation (51). C, The emission spectrums of 5 μM Tranilast fluorescence titrations exhibit the

increasing intensities in the Tranilast fluorescence with increasing concentration of mS100A11 in

micromolar level. D, The curve with fluorescence intensity changes versus the mS100A11 concentrations

by measuring at a wavelength of 333 nm. Dissociation constant (kd) is 11.75 μM calculated from the

equation. The values are the average of three replicates.

FIGURE 2. The structure of Tranilast molecule.

FIGURE 3. Analysis of 1H-15N HSQC titrations of mS100A11 with RAGE V domain. A, Superimposition

of HSQC spectrums of 1.08 mM free 15N-mS100A11 (red) and of 15N-mS100A11 with 0.54 mM unlebeled

RAGE V domain (green). Cross peaks which intensity decreasing (or disappearing) are shown by blue

boxes. B, Bar diagram analysis represents the changes in intensities of cross-peaks of 15N-mS100A11

complex with unlabeled RAGE V domain compared to free mS100A11 versus residue numbers of

mS100A11. The dark red line represents the criterion of selected residues which exhibited greater

decreasing signals (<0.3). The selected residues are shown in red bars. C, Bar diagram analysis represents

the perturbations of cross-peaks of mS100A11 complex with RAGE V domain compared to free mS100A11.

The dark red line represents the criterion of selected residues which exhibited greater perturbations (>0.06).

The selected residues are shown in red bars. Perturbations were calculated by using the equation: combined

shift difference = [(proton shifts)2 + (nitrogen shifts/6.51)2]0.5 (60) D, Selected residues were labeled in red

on the three dimensional structure of homodimeric S100A11 (green) using the PyMOL program.

FIGURE 4. Analysis of 1H-15N HSQC titrations of RAGE V domain in complex with mS100A11. A,

Superimposition of HSQC spectrums of 0.72 mM free 15N-RAGE V domain (red) and 15N-RAGE V domain

complex with 0.72 mM unlebeled mS100A11 (green). More significant signal changes are shown by blue

boxes. B, Bar diagram analysis represents the changes in intensities of cross-peaks of 15N-RAGE V domain

complex with unlabeled mS100A11 compared to free RAGE V domain. The dark red line represents the

criterion of selected residues which exhibited greater decreasing signals (<0.5). The selected residues are

shown in red bars. C, Selected residues were labeled in yellow on the three dimensional structure of RAGE


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V domain (cyan) using the PyMOL program.

FIGURE 5. Binary complex model of S100A11-RAGE V domain determined from the HADDOCK

program. A, The 20-lowest energy backbone structures of S100A11-RAGE V domain binary complex. The

backbone of RAGE V domain and S100A11 are orange and dark blue, respectively. B, The expansion region

was shown as the binding site between S100A11 and RAGE V domain with side chains of selected residues.

The stick structures of resides N53, Q54 and A90 of S100A11 (green) and of residues W61, V63, S65 and

D73 of RAGE V domain (cyan) are shown in red and yellow, respectively.

FIGURE 6. Analysis of 1H-15N HSQC titrations of mS100A11 in complex with Tranilast. A,

Superimposition of 1H-15N HSQC spectrums of 0.5 mM free 15N-mS100A11 (red) and 15N-mS100A11 with

increasing ratios of Tranilast (green, dark blue and purple). More significant signal changes are indicated

by blue boxes. B, Selected residues affected by Tranilast are labeled in red on the three-dimensional

structure of homodimeric S100A11 (green) using the PyMOL program.

FIGURE 7. The binary complex model of S100A11-Tranilast determined from the HADDOCK program.

A, The results of the HADDOCK calculations show the binding interfaces of S100A11 with Tranilast by

overlapping 13 stick structures of Tranilast molecules at both sides of S100A11 homodimer. S100A11

homodimer is colored in green and binding site with Tranilast are labeled in red. B, The expansion region

was shown as the binding interface between S100A11 and Tranilast with side chains of binding residues.

The stick structures of resides N53, L87 and A90 of one of S100A11 monomer (green) are shown in red.

FIGURE 8. Bar diagram of WST-1 assay analysis. The blue bar-height represents the ratio of cell

proliferation compared to the control. The control (LANE 1) is in serum-free condition without exogenous

mS100A11, RAGE V domain and Tranilast. The values are the average of four replicates.

FIGURE 9. Schematic diagram for mechanism of interaction between S100A11 and RAGE. The proposed

mechanism elucidates that the binding of extracellular S100A11 dimer to RAGE would trigger the

homodimerization of two RAGEs and autophosphorylation of their cytoplasmic domains in RAGE. These

result in activation of signal transduction pathways and promotion of the downstream pathways, followed

by cell proliferation.

FIGURE 10. Superimposition of two complex structures (S100A11-RAGE V domain and S100A11-

Tranilast) S100A11-RAGE V domain complex is painted in green. Tranilast molecules are painted in multi-

color. It is clear that Tranilast blocks the interaction between mS100A11 and RAGE V domain.


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Yen Kai Huang, Ruey Hwang Chou and Chin YuAdvanced Glycation End Products (RAGE) V Domain and Inhibits Cell Proliferation

Tranilast Blocks the Interaction between the Protein S100A11 and Receptor for

published online May 12, 2016J. Biol. Chem.

10.1074/jbc.M116.722215Access the most updated version of this article at doi:

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