The effect of Zn2+ on Exopalaemon carinicauda arginine kinase: Computational simulations including unfolding kinetics

Accepted Manuscript Title: The effect of Zn2+ on Exopalaemon carinicauda arginine kinase: computational simulations including unfolding kinetics Author: Yue-Xiu Si Jinhyuk Lee Shang-Jun Yin Meng-Lin Zhang Guo-Ying Qian Yong-Doo Park PII: DOI: Reference:

S1359-5113(14)00598-4 http://dx.doi.org/doi:10.1016/j.procbio.2014.12.007 PRBI 10294

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

25-11-2014 12-12-2014 19-12-2014

Please cite this article as: Si Y-X, Lee J, Yin S-J, Zhang M-L, Qian G-Y, Park Y-D, The effect of Zn2+ on Exopalaemon carinicauda arginine kinase: computational simulations including unfolding kinetics, Process Biochemistry (2014), http://dx.doi.org/10.1016/j.procbio.2014.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The effect of Zn2+ on Exopalaemon carinicauda arginine

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kinase: computational simulations including unfolding

3

kinetics

4

Yue-Xiu Sia,1, Jinhyuk Leeb,c,1, Shang-Jun Yina, Meng-Lin Zhanga, Guo-Ying Qiana,*,

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and Yong-Doo Parka,d,*

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a

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Ningbo 315100, P.R. China

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b

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Biotechnology, Daejeon 305-806, Korea

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College of Biological and Environmental Sciences, Zhejiang Wanli University,

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Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and

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c

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305-350, Korea

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d

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Institute of Tsinghua University, Jiaxing 314006, P. R. China

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Department of Bioinformatics, University of Sciences and Technology, Daejeon

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Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region

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These authors equally contributed to this study.

*

Corresponding authors: Guo-Ying Qian, Tel: +86 57488222298; Fax: +86

57488222298; E-mail address: [email protected]. Yong-Doo Park, Tel:

+86

57488222391;

Fax:

+86

57488222957.

E-mail

address:

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[email protected].

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Abbreviations used: ECAK, arginine kinase from Exopalaemon carinicauda; ANS,

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1-anilinonaphthalene-8-sulfonate; ATP, adenosine triphosphate; MD, molecular

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dynamics.

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Page 1 of 50

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ABSTRACT

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Arginine kinase (AK) plays an important role in the cellular energy metabolism of

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invertebrates. We investigated the effects of Zn2+ on Exopalaemon carinicauda

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arginine kinase (ECAK). Zn2+ conspicuously inactivated the activity of ECAK (IC50 =

6

8.49 ± 0.76 µM), and the double-reciprocal kinetics indicated that Zn2+ induced

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non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed

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that Zn2+ induced tertiary structure changes in ECAK with the exposure of

9

hydrophobic surfaces that directly induced ECAK aggregation. The addition of

10

osmolytes, such as glycine and proline, successfully blocked the ECAK aggregation

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and restored the conformation and activity of ECAK. We measured the ORF gene

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sequence of ECAK using RACE and built a 3D structure of ECAK using homology

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models. Additionally, molecular dynamics (MD) and docking simulations between

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ECAK and Zn2+ have been conducted. The simulation results showed that Zn2+

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blocked the entrance of ATP to the active site, and this result is consistent with the experimental result showing Zn2+-induced inactivation of ECAK. Our study demonstrates the effect of Zn2+ on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding. This

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study might provide important insights into the AK metabolic enzyme of invertebrates

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in marine environments.

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Keywords: arginine kinase; Zn2+; inhibition; aggregation; osmolytes; MD simulation

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1. Introduction

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Phosphagen kinases are a group of highly conserved enzymes that catalyze the

4

reversible transfer of phosphoryl groups from phosphagens; for example, arginine

5

kinase catalyzes the generation of phosphoarginine (ATP: arginine phosphotransferase,

6

EC 2.7.3.3, AK) [1,2]. AK in invertebrates plays a critical role in both the temporal

7

and spatial ATP buffering in cells with the high and fluctuating energy requirements,

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mainly in muscle and nerves, by catalyzing magnesium-dependent reversible

9

phosphorylation involving L-arginine and ATP; this reaction is connected to the

10

regulation of cellular energy homeostasis [3,4]. In general, the depicted structures of

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AKs from different sources showed that the functional N-terminal domain of these

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AKs is the same as that of ATP:guanido phosphotransferase, which has a guanidine

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substrate specificity domain (GS domain), and the same as the ATP-gua Ptrans

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domain responsible for ATP binding (PDB accession numbers: 1BG0, 1M15, 1M80,

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1P50, 1P52, 1RL9, 1SD0, and 2J1Q). Regardless, most AKs are monomers with an average molecular mass of approximately 40 kDa, although marine echinoderms possess a dimeric AK [5-7]. Homologous amino acid sequence alignments of AK suggest that members of the phosphagen kinase family evolved from a common

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ancestor [8-10]. The evolution of a marine organism’s metabolic patterns generally

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leads to further adaptation to the aquatic environment. For instance, hyperosmotic

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treatments led to a reduction in AK flux through an increase in the concentration of

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perturbing inorganic ions, while hypoosmotic treatments led to an enhanced AK flux

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Page 3 of 50

[11]. Some marine organisms utilize inorganic metal ions and amino acids as osmotic

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solutes to maintain cell volume to adapt to the ionic strength of seawater [12,13].

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However, the increasing pollution from metal ions in seawater often leads to

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intracellular accumulation in marine organisms. Thus, it is interesting to investigate

5

the effects of metal ions and osmolytes on AK structure and function.

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Zn2+ is an essential component of many enzymes and plays various roles in

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metabolism and diseases [14-18]. Thus, Zn2+ contributes to the regulation of many

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biological processes, and adequate Zn2+ is necessary for maintaining the homeostasis

9

of many organisms. However, excessive Zn2+ can be toxic to organisms in different

10

ways [19,20]. It is evident that Zn2+ can directly induce hydrophobic exposure,

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unfolding, and aggregation in a broad variety of proteins and metallic-/non-metallic

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enzymes, including creatine kinase, AK and aminoacylase [21-26]. The roles of Zn2+

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in ocean invertebrates have not been well elucidated. It has been found that the Zn2+

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concentrations in the tissues of aquatic organisms, including marine invertebrates, are

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usually far in excess of that required for normal metabolism [27-29]. Much of the excess Zn2+ is bound to macromolecules or present as insoluble metal inclusions in tissues [30]. For this reason, it is necessary to study the roles of Zn2+ and the effect of Zn2+ on enzymes in aquatic organisms.

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To date, several AKs from different types of shrimp have been purified and

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characterized [31-33]. Exopalaemon carinicauda is a white prawn that is important

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for commercial marine products. Recently, studies regarding immune genes in E.

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carinicauda has been reported [34-36]. Investigating the energy-related enzymatic

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Page 4 of 50

properties of E. carinicauda might provide valuable information on the mechanisms

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involved in the adaptation to marine environments. In this work, we investigated the

3

effects of Zn2+ on AK from E. carinicauda and the role of osmolytes with respect to

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functional and structural changes and combined these studies with computational

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simulations. This study provides insight into the unfolding responses of ECAK

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induced by Zn2+ and might reveal the functional role of osmolytes, such as glycine

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and proline, in preventing aggregation.

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2. Materials and Methods

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2.1. Materials

ATP, arginine, magnesium acetate, thymol blue, zinc acetate dihydrate, glycine,

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proline, and 1-anilinonaphthalene-8-sulfonate were purchased from Sigma-Aldrich.

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All other chemicals were locally obtained and of the highest analytical grade.

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2.2. ECAK purification

ECAK was purified from muscle samples using a Cellulose DE-52 ion-exchange

chromatograph and a Hiprep 26/60 Sephacryl S-200 HR gel filtration chromatograph

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with 20 mM Tris-acetic acid buffer (pH 8.0). The purified ECAK was shown to be

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homogeneous using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE).

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2.3. ECAK activity assay AK activity was measured by monitoring the proton generation during the reaction

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of ATP and arginine with thymol blue at 20°C, as previously described [37,38]. The

4

substrate mix was composed of 5.7 mM arginine, 5 mM ATP, 6.6 mM magnesium

5

acetate, and 0.015% thymol blue, pH 8.0. The reaction volume was 1 ml, and 10 µl of

6

enzyme solution was added to the substrate system. Absorption was recorded at 575

7

nm using a Shimadzu UV-1800 spectrophotometer.

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2.4. Kinetic analysis

To evaluate the inactivation kinetics and rate constants, the transition free energy

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was calculated based on methods described in a previous report with slight

12

modifications [39]. The transition free energy change per second is given by ΔΔG˚ =

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–RTlnk. The data were calculated from semi-logarithmic plots, and k is the time

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constant for the major phase of the inactivation reaction.

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For a general analysis of non-competitive inhibition, the Lineweaver-Burk equation

can be written in double-reciprocal form:

1 Km [I] 1 1 [I]  (1  )  (1  ) v Vmax K i [S] Vmax Ki

(1)

Secondary plots can be constructed from Y-intercept 

1 Vmax



1 [ I] K iVmax

(2)

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The Ki, Km, and Vmax values can be derived from these two equations. The secondary

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replot of Y-intercept vs. [I] is linearly fitted assuming a single inhibition site or a

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Page 6 of 50

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single class of inhibition sites.

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2.5. Protein folding For the unfolding experiment, ECAK was dissolved in buffer with different

5

concentrations of Zn2+ for 2 hours at 20°C. The refolding experiment was performed

6

by adding different concentrations of osmolytes to ECAK that had been previously

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treated with 1 mM Zn2+. Fluorescence emission spectra were measured using an

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F-4500 spectrofluorometer (Hitachi, Japan) and a 1-cm path-length cuvette. The

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excitation and emission wavelengths were 280 nm and 300-400 nm, respectively.

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ANS was used to probe the hydrophobic surface, and a 50-fold excess concentration

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of ANS was added to the samples for 30 min in the dark. Fluorescence was measured

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using a 1-cm path-length cuvette and excitation and emission wavelengths of 380 nm

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and 400-600 nm, respectively. All spectra were collected at 20°C in 20 mM Tris-acetic

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acid buffer (pH 8.0).

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2.6. ECAK aggregation measurement induced by Zn2+ The aggregation of ECAK induced by Zn2+ was monitored by recording the

absorbance at a wavelength of 400 nm in a Shimadzu UV-1800 spectrophotometer

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using a 1-cm path-length cuvette. To measure the effect of Zn2+, the aggregation time

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course was measured at different Zn2+ concentrations. The final enzyme concentration

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was 20 μM, and the recording time was 7200 s.

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For kinetic analysis of ECAK aggregation, the following equations, which are

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Page 7 of 50

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outlined in previous reports [40,41], were applied: ΔAG = AG − AGt

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(3)

where AG is the absorbance at the end of the aggregation reaction before reaching

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the precipitation state, and AGt is the absorbance at time t during aggregation. The

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experimental data were fitted to first-order expressions:

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ΔAG = exp(−kAG/t)

(4)

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ΔAG = P1exp(−kAG1/t) + P2exp(−kAG2/t) + P3exp(−kAG3/t)

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(5)

where kAG is the rate constant for a monophasic reaction (Equation (4)). P1, P2, and P3

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indicate the fractions reacting with the rate constants kAG1, kAG2, and kAG3, respectively.

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The change in the transition free energy during aggregation in the presence of an

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additive is expressed as:

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2.7. Statistical analysis

(6)

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ΔΔGAG = RTln (kAG,none/kAG,additive)

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All statistical data were analyzed using the General Linear Model (GLM)

Procedure of SAS 8.0. Differences among means were tested using Duncan’s new multiple range test. A significant difference among groups was shown as P<0.05.

2.8. cDNA cloning of ECAK and RACE

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Total RNA was isolated from the tissue samples (weight: 50 mg) using Trizol

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reagent (Invitrogen, Japan) following the manufacturer’s protocol. The integrity was

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assessed by analysis on a 1.5% agarose gel, and the concentrations were determined

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Page 8 of 50

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using a spectrophotometer. cDNA was synthesized from 2 μg of total RNA using

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M-MLV reverse transcriptase (Takara, Japan) at 42 °C for 1 h with Oligo dT primer

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following the protocol of the manufacturer. Oligonucleotide primers for the

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amplification

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CAACCAGCACTGYGGCATCTAC

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TGAARAGRAAGTGGTCATCRAC. The PCR products were separated using a 1.2%

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agarose gel and then purified using a PCR purification kit. The purified PCR product

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was ligated with the pMD18-T vector (Takara, Japan) and transformed into competent

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Escherichia coli cells. Positive clones were sequenced. The sequences were analyzed

cDNA

were

designed and

as

sense

ip t

ECAK

antisense

for

similarities

to

other

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(http://www.ncbi.nim.nih.gov/).

known

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sequences

using

BLAST

The rapid amplification of cDNA end (RACE) technique was used to extend the

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cDNA end of the ECAK sequence, including the 3’- and 5’-UTR, in accordance with

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the manufacturer’s instructions for the 3’-Full RACE Kit and the 5’-Full RACE Kit

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(Takara, Japan). For 3’ RACE, 2 μL of RT product was amplified using PCR with nested forward primers (3’ RACE1 as AGCATTGATGGCTTCGGTCTC and 3’ RACE2 as TGGCATCACCAAGGAACAGC) and GCTGTCAACGATACGCTACG TAACGGCATGACAGTGTTTTTTTTTTTTTTTTTT as an adaptor primer. After an

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initial 3 min denaturation at 94 °C, 30 cycles were performed as follows: 30 s

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denaturation at 94 °C, 30 s annealing at 57 °C and 2 min elongation at 72 °C followed

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by a final 10 min at 72 °C. For 5’ RACE, the first strand of cDNA obtained was tailed

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with poly (C) at the 5’ ends using terminal deoxynucleotidyl transferase (TdT) (Takara,

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Page 9 of 50

Japan). Outer PCR was performed with TTCCTTGGTGATGCCAGGGGAGAGAC

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as the 5’ RACE1 primer and GCTGTCAACGATACGCTACGTAAC as the outer

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primer. The PCR temperature profile began with an initial 3 min denaturation at 94 °C,

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and 30 amplification cycles were performed as follows: 30 s denaturation at 94 °C, 30

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s annealing at 65 °C and 2 min elongation at 72 °C, and a final elongation at 72 °C for

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10 min. Inner PCR was performed with GGACTTCGGAGTTGATGTTGCCCTTG as

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the 5’ RACE2 primer and GCTACGTAACGGCATGACAGTG as the inner primer.

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PCR was performed under hot-start conditions (94 °C, 3 min) with TaKaRa LA Taq

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(Takara, Japan) for 30 cycles of 94 °C (30 s), 65 °C (30 s), and 72 °C (2 min), and

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then 10 min at 72 °C. Finally, the products were gel-purified, cloned and sequenced.

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2.9. Computational simulations of ECAK

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Because the ECAK protein structure has not been determined, the PQR-SA

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(pseudo-quadratic restraints simulated annealing) homology modeling method [42]

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was used to generate the structure. PQR-SA generated 40 conformations by performing simulated annealing with different initial velocities. The STAP (statistical torsion angle potential) energy potential [43] was used to develop a highly protein-like structure. Of the resulting conformations, the best conformation with the

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lowest energy structure was chosen. To validate that the structure is well-packed,

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various protein structure properties (the radius of gyration (Rg), distance heat-map,

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and protein-like scores) were measured. All of the calculations were performed using

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CHARMM (chemistry at Harvard macromolecular mechanics) [44]. A plausible

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Page 10 of 50

active site of the homology protein structure was predicted using DPSP (Dockable

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Pocket Site Prediction). This method is based on the template structures and the

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ligands bound on these structures. Two molecular dynamics (MD) simulations with

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and without Zn2+ were performed using the homology model. Nine Zn2+ were

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randomly distributed to approximate a concentration of 10 mM. A periodic box with a

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dimension of 110 Å was used for prevent the ions from drifting away. The homology

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protein structure was positioned at the origin. A short minimization and equilibrium

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dynamics were performed. Afterward, 10 ns (nanosecond) of production MD was run

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and trajectories were every 10 ps (picosecond) for further analyses. For trajectory

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analyses, the root mean square deviation (RMSD) from the initial structure and the

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root mean square fluctuation (RMSF) as a function of residue number and the number

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of bound Zn2+ were measured. Two final structures were used to measure the

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secondary structure ratio (alpha/beta/coil) using DSSP (Dictionary of protein

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Secondary Structure: Pattern recognition of hydrogen-bonded and geometrical

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features) program [45].

3. Results

3.1. Effect of Zn2+ on ECAK activity

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The purified ECAK was assayed after incubation with different concentrations of

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Zn2+ for 2 h. Zn2+ inhibited ECAK in a dose-dependent manner (Fig. 1). The ECAK

22

activity was almost abolished when the Zn2+ concentration was 0.05 mM (Fig. 1A).

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Page 11 of 50

The IC50 value was measured to be 8.49 ± 0.76 µM (n = 3). When Zn2+ was absent in

2

the assay system where the sample enzyme could be refolded due to the dilution effect,

3

the IC50 value was measured to be 9.26 ± 0.80 µM (n = 3), which was almost exactly

4

the same as the result of Fig. 1A. The results showed that ECAK was unfolded by

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Zn2+ completely, and the unfolded ECAK could not be refolded using a simple diluted

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technique (Fig. 1B). The results also implied that ECAK undergoes serious

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conformational changes due to Zn2+.

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(Insert Fig. 1)

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3.2. Noncompetitive inhibition induced by Zn2+

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The kinetics of the enzyme was studied in the presence of Zn2+ using

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double-reciprocal Lineweaver-Burk plots. The results showed apparent Vmax changes,

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which indicated that Zn2+ induced non-competitive inhibition of arginine (Fig. 2A)

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and ATP (Fig. 3A). The secondary replot of Y-intercept (1/Vmax) vs. [Zn2+] was

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linearly fitted (Fig. 2B and Fig. 3B), showing that Zn2+ contains a single class of inhibition site for ECAK. The Ki values were calculated to be 5.14 ± 0.68 µM for arginine and 7.43 ± 1.21 mM for ATP. Furthermore, the kinetic parameters for ECAK were measured to be Km = 0.34 ± 0.02 mM and Vmax = 67.14 ± 7.61 μmoles/min for arginine and Km = 3.23 ± 0.24 mM and Vmax = 71.14 ± 5.83 μmoles/min for ATP.

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(Insert Fig. 2)

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(Insert Fig. 3)

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The kinetic analyses of ECAK inactivation indicated that Zn2+ might decrease the

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Page 12 of 50

1

activity of ECAK by changing its structure. To confirm our thoughts, we next

2

measured the tertiary structural change of ECAK in the presence of Zn2+.

3

3.3. Zn2+-induced tertiary structural change of ECAK

ip t

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The spectroscopic spectra were obtained to elucidate the effects of Zn2+ on the

6

ECAK tertiary structure. Intrinsic fluorescence was used to monitor the ECAK

7

tertiary conformation changes at different Zn2+ concentrations (Fig. 4). The maximum

8

emission peak wavelength of ECAK was 330 nm. The maximum emission

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wavelength was slightly red-shifted from 330 to 332 nm with increasing Zn2+

10

concentration (Fig. 4A), whereas the intensity of the spectra was not significantly

11

changed by Zn2+ (Fig. 4B). This finding indicated that Zn2+ did not modulate the

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tertiary structure of ECAK significantly but its binding slightly loosened the structure

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of ECAK.

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(Insert Fig. 4)

However, this result alone is not sufficient to explain the complete inactivation of

ECAK and the unfolded state that is not spontaneously refolded via simple dilution. We hypothesized that as the Zn2+ concentration increased, the exposure of the ECAK hydrophobic environment for the fluorescent chromophore caused by Zn2+ binding

19

could directly modulate the enzyme activity. Thus, the effect of Zn2+ on the exposure

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of ECAK hydrophobic surfaces was probed through measurements of ANS-binding

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fluorescence spectra (Fig. 5). The results showed that the binding of Zn2+ to the

22

enzyme led to a conspicuous increase in the ANS fluorescence in a dose-dependent

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Page 13 of 50

manner, indicating that hydrophobic disruption of ECAK occurred as a result of Zn2+

2

binding. Compared with that for the native state of ECAK, the ANS-fluorescence

3

intensity was significantly increased, indicating that Zn2+ directly induced tertiary

4

structural changes, mainly in the hydrophobic surfaces of ECAK rather than the

5

overall structure, and this change directly caused the loss of activity of the enzyme.

6

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(Insert Fig. 5)

The highly exposed hydrophobic surfaces of the enzyme could be prone to

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misfolding; in particular, they could induce aggregation. We next tested the

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Zn2+-mediated ECAK aggregation phenomenon, which is frequently associated with the folding pathway, and observed that it competed with correct folding.

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3.4. ECAK aggregation induced by Zn2+

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We examined the aggregation process of ECAK when it was incubated with Zn2+

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under suitable conditions (Fig. 6). The ECAK aggregation was dependent on Zn2+; as

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the Zn2+ concentration increased from 0.4 to 2.0 mM, the aggregation increased remarkably (Fig. 6A). Kinetic analysis based on Equations (3) to (5) showed that the aggregation underwent a multi-phasic kinetic process, that is, bi- and tri-phase processes (Figs. 6B to 6F). The rate constants for the ECAK aggregation induced by

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Zn2+ are summarized in Table 1. The rate of aggregation was sensitively modulated by

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the Zn2+ concentration.

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(Insert Fig. 6)

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(Insert Table 1)

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Page 14 of 50

The values shown in Table 1 correspond to a first-order kinetic reaction. The partial

2

aggregation of ECAK was a bi-phasic process at a relatively high concentration of

3

Zn2+ (0.4 to 0.6 mM) compared with the concentration for the activity change, i.e., the

4

concentration where ECAK was effectively fully inactivated (the remaining activity

5

was less than 5% at 0.05 mM). The ECAK aggregation underwent a tri-phasic process

6

at higher concentrations of Zn2+. The shift in the aggregation process from bi-phasic

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to tri-phasic implies that ECAK transiently passed through intermediates until it

8

reached a completely misfolded state. From the evaluated rate constants, which were

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calculated from the semi-logarithmic plots, the change in the transition free energy

10

(ΔΔG˚) was calculated based on Equation (6). The aggregation process occurred as a

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result of a decrease in the change in the transition free energy (ΔΔG˚), which occurred

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in a Zn2+ concentration-dependent manner.

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3.5. Effect of osmolytes on the inactivation of ECAK by Zn2+

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Next, we measured the protective role of some osmolytes as folding chaperones

that promote the correct folding of ECAK to overcome the Zn2+-induced aggregation. To identify effective folding chaperones for ECAK, we tested several known osmolytes, such as glycerol, sucrose, glycine, proline and liquaemin, which were

19

previously found to be effective folding aids. The results showed that of these

20

osmolytes, two amino acids (glycine and proline) were found to be the most effective

21

chaperones for ECAK.

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(Insert Fig. 7)

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Page 15 of 50

After the reaction of ECAK with 0.1 mM Zn2+ (at this condition, ECAK was

2

completely inactivated), the inactivated enzyme was incubated with different

3

concentrations of glycine for 2 h, and the activity of the sample was measured (Fig.

4

7A). Similarly, ECAK that had been inactivated by Zn2+ was incubated with different

5

concentrations of proline (Fig. 7B). The activity of ECAK was significantly recovered

6

with increasing glycine and proline concentrations in a dose-dependent manner. When

7

the concentration of glycine was 50 mM, 93% of the activity of ECAK was recovered.

8

Similarly, approximately 85% of the ECAK reactivity was obtained after incubation

9

with proline at a concentration of 120 mM. These two osmolytes were therefore found

10

to be effective in recovering ECAK activity. In addition to these two osmolytes,

11

liquaemin was partially effective for the Zn2+-treated ECAK (Fig. 7C). When the

12

inactivated enzyme sample was incubated with 1.0 mg/mL liquaemin, 61% of the

13

activity was recovered. Glycerol and sucrose were not effective at protecting ECAK

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from Zn2+-induced inactivation.

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3.6. Effect of osmolytes on the recovery of the tertiary structure of ECAK The effect of the osmolytes on the conformational structure of ECAK was assessed

by monitoring the ANS-binding fluorescence of Zn2+-treated ECAK. Glycine did not

19

affect the exposure of the hydrophobic surfaces of ECAK that was induced by 0.4

20

mM Zn2+, proline slightly suppressed the exposure of the hydrophobic surfaces of

21

ECAK,

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hydrophobicity (Fig. 8). These results indicated that the restoration of activity by

and

liquaemin

significantly

suppressed

the

Zn2+-induced

ECAK

16

Page 16 of 50

1

liquaemin could be mainly dependent on the liquaemin-induced restoration of the

2

hydrophobic surface of ECAK induced.

3 4

3.7. Protective effect of osmolytes on the ECAK aggregation

cr

5

ip t

(Insert Fig. 8)

We next tested the effects of glycine, proline and liquaemin on the aggregation of

7

ECAK in the presence of Zn2+. The aggregation of ECAK induced by Zn2+ was

8

gradually and effectively blocked by the addition of glycine, proline, or liquaemin

9

(Fig. 9). Glycine, proline and liquaemin could completely prevent ECAK aggregation

10

in the conditions with 2.0 mM Zn2+ and either 5 mM glycine (Fig. 9A) or 90 mM

11

proline (Fig. 9B). The greatest effects were found for 0.6 mg/ml liquaemin (Fig. 9C).

12

These osmolytes acted as stabilizers for the reactivation of ECAK and effectively

13

suppressed aggregation.

15 16 17 18

an

M

d

te

Ac ce p

14

us

6

(Insert Fig. 9)

3.8. RACE and the gene cloning of ECAK The 1068-bp ECAK fragment was amplified with degenerate primers. ECAK

gene-specific primers, 3’RACE1 and 3’RACE2, were designed to clone the 3’ end of

19

ECAK cDNA, and a 922-bp fragment was amplified using the 3’ RACE technique.

20

We performed 5’RACE-PCR with the specific primers 5’RACE1 and 5’RACE2 and

21

amplified a 430-bp fragment. A 1685-bp nucleotide sequence representing the

22

complete cDNA sequence of the ECAK gene was obtained by cluster analysis of the

17

Page 17 of 50

1

above sequences. The complete cDNA of the ECAK gene was deposited in GenBank

2

under accession number 1769040.

4

3.9. Homology modeling and MD simulations of ECAK and Zn2+.

ip t

3

The generated homology model is shown in Fig. 10A. We generated 40

6

conformations and picked the lowest energy conformations. Seven template protein

7

structures were used for the homology structure modeling (1qh4, 3l2f, 1m15, 3jpz,

8

3fmb, 3f3f, and 2rno) from the Protein Data Bank. The best template structure is 1qh4

9

with a sequence identity of 0.43. The structure 1qh4 originates from the crystal

10

structure of chicken brain-type creatine kinase. To validate the chosen homology

11

protein structure, the radius of gyration (Rg) and the distance heat-map were

12

calculated (Figs. 10B and C). The calculated Rg is 20.2 Å. The Rg value is

13

appropriate because the Rg falls on the fitting line of the graph (blue line in Fig. 10B).

14

The distance heat-map shows the typical arginine kinase pattern (Fig. 10C).

16 17 18

us

an

M

d

te

Ac ce p

15

cr

5

(Insert Fig. 10)

With the homology protein structure, the ADP binding site (ADP is an analogue of

ATP) was predicted using the DPSP server (Fig. 11A). The best ADP ligand score was located in pocket number 13, which consists of 52 residues (red and yellow spheres in

19

Fig. 11) and has a volume of 3,483 Å3. The predicted ADP-interacting residues consist

20

of 16 residues (yellow spheres in Fig. 11A) with a DPSP score of 0.3 (circle in the

21

heat-map) as shown in Fig. 11B.

22

(Insert Fig. 11)

18

Page 18 of 50

With the generated homology ECAK structure, 10 ns MD simulations were

2

performed with/without Zn2+. Two MD simulation stabilities were checked. The

3

RMSD plots of both simulations were stabilized after ~ 3 ns with an RMSD of 3.5 Å

4

relative to the initial structure (Fig. 12A). Nine zinc ions were bound to ECAK in ~

5

1.5 ns (Fig. 12B). To determine where Zn2+ was bound, the RMSF was calculated

6

with respect to the residue numbers (Fig. 12C). The final structure contains 58

7

residues that bind to Zn2+ (red box symbols, Fig. 12C), 16 residues that bind to ADP

8

(16 total residues; blue and red boxes in Fig. 12C and yellow spheres in Fig. 11A),

9

and three of these residues bind to both (THR304, ARG305, and ASP317; see 3D

10

visualization in Fig. 11B). This finding indicates that Zn2+ can block the entrance of

11

the ADP-binding site of ECAK. Two final structures are structurally compared in Fig.

12

13. The two structures were superimposed, and the RMSD of the alpha carbons was

13

2.43 Å. The secondary structure with Zn2+ was slightly changed from that of the

14

structure without Zn2+ (Fig. 13B). The helix/sheet/coil secondary structure ratios are

16 17 18

cr

us

an

M

d

te

Ac ce p

15

ip t

1

same (35%/15%/50%). However, the locations of the three secondary structures changed slightly.

(Insert Fig. 12) (Insert Fig. 13)

19 20

4. Discussion

21 22

The ECAK gene and the full ORF sequence using the RACE technique were firstly

19

Page 19 of 50

1

revealed in this study. On the basis of the gene sequence results, we set up a

2

reasonable 3D structure of ECAK and conducted serial computational simulations for

3

the ligand binding of Zn2+. Zn2+ markedly inhibited ECAK in a dose-dependent manner and induced

5

substantial hydrophobic exposure that was accompanied by the aggregation of ECAK

6

under proper conditions. We confirmed that the conformational status of the overall

7

ECAK structure was partially disrupted by Zn2+ binding, but the hydrophobicity of the

8

enzyme was conspicuously increased, which resulted in the misfolding of ECAK.

9

Interestingly, several osmolytes such as glycine, proline, and liquaemin were shown to

10

be folding aids for ECAK, and these osmolytes effectively restore the Zn2+-treated

11

ECAK and successfully block the aggregation of ECAK in the presence of Zn2+.

M

an

us

cr

ip t

4

Because the range of Zn2+ concentrations used in the present study was comparable

13

to physiological concentrations, our results help to elucidate the role of Zn2+ in ECAK

14

as a negative activity regulator. To clarify the possible physiological roles of Zn2+ and

16 17 18

te

Ac ce p

15

d

12

osmolytes in ECAK and their relationship with the folding of ECAK, including the aggregation phenomenon, more systemic sequential studies should be conducted. We found that among the osmolytes tested, glycine, proline and liquaemin not only

prevented aggregation but also rescued the activity and structure of ECAK that were

19

destroyed due to the unfolding induced by Zn2+, and these results suggest that

20

osmolytes protect metabolic enzymes from unfavorable misfolding, although we still

21

do not know whether surplus Zn2+ induces the aggregation of ECAK under in vivo

22

conditions.

20

Page 20 of 50

The multi-phase process observed during aggregation implied that transient

2

intermediates of ECAK existed during the Zn2+-induced unfolding. The Zn2+-induced

3

aggregation was most likely caused by the accumulation of such intermediates with

4

increasing Zn2+ concentrations. After ECAK passed through the intermediate states

5

and reached a completely unfolded state, it was easily misfolded, resulting in

6

aggregation. Osmolytes, such as glycine, proline, and liquaemin, may competitively

7

stabilize the folding intermediates of ECAK at this turning point for producing

8

aggregation. Proline and liquaemin stabilized the hydrophobic surfaces of ECAK

9

intermediates and simultaneously, they could restore the catalytic function of the

cr

us

an

enzyme.

M

10

ip t

1

Computational simulation results indicated that a Zn2+ is found in the active site of

12

ECAK. The neighboring residues that interacted with the Zn2+ shared common

13

residues (THR304, ARG305, and ASP317) with creatine/ATP binding sites. This

14

finding may explain the inactivation mechanism shown in the experiment in which

16 17 18

te

Ac ce p

15

d

11

Zn2+ acted as a noncompetitive inhibitor. In conclusion, we investigated the inhibitory effects of Zn2+ on ECAK. Our

findings provide concrete evidence of the toxicity of Zn2+ in the context of metabolic enzymes and ECAK or a putative role as a negative regulator. Our results provide new

19

insight into the relationship between Zn2+ and ECAK, including the functional role of

20

osmolytes in preventing aggregation; thus, our results elucidate the mechanisms

21

underlying Zn2+-related functions in marine invertebrates.

22

21

Page 21 of 50

1

Acknowledgments

2

This study was supported by the Zhejiang Provincial Top Key Discipline of

4

Biological Engineering (No. CX2014014 and KF20140010) and the National

5

Undergraduate Training Programs for Innovation and Entrepreneurship (No.

6

201410876001). Dr. Guo-Ying Qian was supported by the Innovation Team Project of

7

Ningbo Municipal Science and Technology Bureau (No. 2012B82016). Shang-Jun

8

Yin was supported by a grant from the 624 project supported by the Zhejiang Leading

9

Team of Science and Technology Innovation (Team No. 2010R50019). Dr. Jinhyuk

10

Lee was supported by a grant from the Korea Research Institute of Bioscience and

11

Biotechnology (KRIBB) Research Initiative Program, the Korean Ministry of

12

Education, Science and Technology (MEST) (2012R1A1A2002676), and the Pioneer

13

Research Center Program through the National Research Foundation of Korea funded

14

by the Ministry of Science, ICT & Future Planning (2013M3C1A3064780). Dr.

16 17 18

cr

us

an

M

d

te

Ac ce p

15

ip t

3

Yong-Doo Park was supported by a grant from the Zhejiang Provincial Natural Science Foundation of China, “Towards studying the function of C3dg protein and elucidating its role in the pathogenesis of atopic dermatitis” (Grant No. LY14H110001).

19 20

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[2] Buttlaire DH, Cohn M. Interaction of manganous ion, substrates, and anions with

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[3] Shofer SL, Willis JA, Tjeerdema RS. Effects of hypoxia and toxicant exposure on

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[10] Azzi A, Clark SA, Ellington WR, Chapman MS. The role of phosphagen

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specificity loops in arginine kinase. Protein Sci 2004;13:575-85.

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muscle of the blue crab Callinectes sapidus. J Exp Biol 2002;205:1775-85.

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bind zinc: an examination of innovative approaches to improved metalloproteinase

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inhibition. Biochim Biophys Acta 2010;1803:72-94. [15] Fukada T, Yamasaki S, Nishida K, Murakami M, Hirano T. Zinc homeostasis and signaling in health and diseases: Zinc signaling. J Biol Inorg Chem 2011;16:1123-34. [16] Sensi SL, Paoletti P, Koh JY, Aizenman E, Bush AI, Hershfinkel M. The

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[17] Prasad AS. Discovery of human zinc deficiency: its impact on human health and

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[18] Maret W. Zinc biochemistry: from a single zinc enzyme to a key element of life.

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β-amyloid structure and dynamics: implications for Aβ aggregation. Biophys J

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Zinc ions induce the unfolding and self-association of boar spermadhesin PSP-I, a

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protein with a single CUB domain architecture, and promote its binding to heparin.

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Biochemistry 2006;45:8227-35.

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[23] Tong X, Zeng X, Zhou HM. Effects of zinc on creatine kinase: activity changes,

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conformational changes, and aggregation. J Protein Chem 2000;19:553-62.

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effect of Zn2+ on Euphausia superba arginine kinase: Unfolding and aggregation

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studies. Process Biochemistry 2014;49:821-9.

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[26] Liu T, Wang X. Zinc induces unfolding and aggregation of dimeric arginine

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kinase by trapping reversible unfolding intermediate. Acta Biochim Biophys Sin

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organs of adult Fundulus heteroclitus after waterborne zinc exposure in freshwater

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and saltwater. Arch Environ Contam Toxicol 2012;63:544-53.

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León-Hill CP, González-Farías F. Accumulation and regulation effects from the metal

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mixture of Zn, Pb, and Cd in the tropical shrimp Penaeus vannamei. Biol Trace Elem

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commercially important finfish and shellfish of the River Ganga. Environ Monit

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Assess 2012;184:2219-30.

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Contaminant Hazard Reviews Report 1993;26:1-126.

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[31] Ma FF, Liu QH, Guan GK, Li C, Huang J. Arginine kinase of Litopenaeus

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vannamei involved in white spot syndrome virus infection. Gene 2014 10;539:99-106. [32] López-Zavala AA, García-Orozco KD, Carrasco-Miranda JS, Sugich-Miranda R, Velázquez-Contreras EF, Criscitiello MF, Brieba LG, Rudiño-Piñera E, Sotelo-Mundo RR. Crystal structure of shrimp arginine kinase in binary complex with arginine-a

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molecular view of the phosphagen precursor binding to the enzyme. J Bioenerg

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Biomembr 2013;45:511-8.

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from a swimming crab, Portunus trituberculatus. Mol Biol Rep 2012;39:4879-88.

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tag (EST) analysis of hemocytes in the ridgetail white prawn Exopalaemon

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carinicauda. Fish Shellfish Immunol 2013;34:173-82.

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[35] Duan Y, Liu P, Li J, Wang Y, Li J, Chen P. Molecular responses of calreticulin

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gene to Vibrio anguillarum and WSSV challenge in the ridgetail white prawn

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Exopalaemon carinicauda. Fish Shellfish Immunol 2014;36:164-71.

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in response to Vibrio anguillarum and WSSV challenge. Fish Shellfish Immunol

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2013;35:661-70.

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refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus

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Chinensis: the solubilization of aggregate and refolding in detergent solutions.

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Biochem Cell Biol 2005;83:140-6.

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[38] Yu Z, Pan J, Zhou HM. A direct continuous pH-spectrophotometric assay for arginine kinase activity. Protein Pept Lett 2002;9:545-52. [39] Tams JW, Welinder KG. Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on

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these processes. Biochemistry 1996;35:7573-9.

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[40] Gou L, Lü ZR, Park D, Oh SH, Shi L, Park SJ, Bhak J, Park YD, Ren ZL, Zou F.

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The effect of histidine residue modification on tyrosinase activity and conformation:

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inhibition

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2

[41] Fang NY, Lee J, Yin SJ, Wang W, Wang ZJ, Yang JM, Qian GY, Si YX, Park YD.

3

Effects of osmolytes on arginine kinase from Euphausia superba: a study on thermal

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denaturation and aggregation. Process Biochem 2014;49:936-47.

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[42] Kim TR, Oh S, Yang JS, Lee S, Shin S, Lee J. A simplified homology-model

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builder toward highly protein-like structures: an inspection of restraining potentials. J

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Comput Chem 2012;33:1927-35.

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[43] Kim TR, Yang JS, Shin S, Lee J. Statistical torsion angle potential (STAP) energy

9

functions for protein structure modeling: a bicubic interpolation approach. Proteins

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2013;81:1156-65.

11

[44] Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B,

12

Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR,

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Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J,

14

Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable

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10

RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M. CHARMM: the biomolecular simulation program. J Comput Chem 2009;30:1545-614. [45] Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition

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1983;22:2577-637.

20 21

28

Page 28 of 50

1

Table 1 Aggregation rate constants for ECAK in the presence of various

2

concentrations of Zn2+.

3

kAG1

kAG2

kAG3

change (kJ/mol·s-1)

0.4

1.77

0.18

-

15.70

0.5

1.94

0.16

-

0.6

2.58

0.13

-

0.75

4.62

1.04

0.15

1.0

13.41

1.19

5

rate constants for the three phases.

an

us

15.47

0.18

14.77 13.32 10.68

M

a

cr

Zn2+ (mM)

4

te

d

Data were calculated as shown in Fig. 6, where kAG1, kAG2 and kAG3 are the first-order

Ac ce p

6

Transition free energy

ip t

Aggregation rate constants (×10-3 s-1) a

29

Page 29 of 50

1

Figure Legends

2

Fig. 1. The effect of Zn2+ on the activity of ECAK.

4

ECAK was incubated with different Zn2+ concentrations at 20°C for 2 h in 20 mM

5

Tris-acetic acid buffer (pH 8.0) and then added to the assay system with the

6

corresponding Zn2+ concentrations (A) or without Zn2+ (B). The final concentration

7

for the enzyme was 0.05 μM. Data are presented as the means (n=3).

us

cr

ip t

3

9

an

8

Fig. 2. Lineweaver-Burk plot for arginine in the presence of Zn2+. (A) The ATP concentration was 5 mM, and the Zn2+ concentrations were 0 (■), 0.01

11

(●), 0.015 (▲), 0.02 (▼) and 0.025 (◄) mM. The final concentration of the enzyme

12

was 0.05 μM. (B) Secondary replot for arginine. Data were collected from (A).

15 16 17 18

d

te

14

Fig. 3. Lineweaver-Burk plot for ATP in the presence of Zn2+.

Ac ce p

13

M

10

(A) The arginine concentration was 5.7 mM, and the Zn2+ concentrations were 0 (■), 0.01 (●), 0.015 (▲) and 0.02 (▼) mM. The final concentration of the enzyme was 0.05 μM. (B) Secondary replot for ATP. Data were collected from (A).

19

Fig. 4. Intrinsic fluorescence changes in ECAK in the presence of Zn2+.

20

The fluorescence emission spectra were measured with excitation at 280 nm and

21

emission in the range of 300-400 nm after the incubation of ECAK with different

22

concentrations of Zn2+ at 20°C for 2 h. The final concentration of the enzyme was 3.5

30

Page 30 of 50

1

μM. (A) Plot of the maximum peak wavelength versus [Zn2+]. (B) Plot of the

2

maximum fluorescence intensity versus [Zn2+].

3

Fig. 5. Changes in the ANS-binding fluorescence of ECAK induced by Zn2+.

5

(A) ECAK was incubated with different concentrations of Zn2+ at 20°C for 2 h and

6

then with ANS (40 µM) for 45 min to label the hydrophobic surface of ECAK prior to

7

measurement. The final concentration of the enzyme was 3.5 μM. Fluorescence was

8

measured with excitation at 390 nm and emission in the range of 420-600 nm. The

9

labels 1 to 9 indicate 0, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5 mM Zn2+,

10

respectively. (B) Maximum ANS-fluorescence intensity versus [Zn2+]. The data were

11

obtained from (A).

cr

us

an

M

d

12

ip t

4

Fig. 6. ECAK aggregation induced by Zn2+ and kinetic analysis.

14

(A) The final concentrations of Zn2+ (labeled 1-7) were 0, 0.4, 0.5, 0.6, 0.75, 1.0 and

16 17 18 19

Ac ce p

15

te

13

2.0 mM, respectively. The treatment temperature was 20°C, and the final concentration of ECAK was 20 μM. (B-F) Kinetic analysis of ECAK aggregation. The concentrations of Zn2+ were 0.4 mM (B), 0.5 mM (C), 0.6 mM (D), 0.75 mM (E), and 1.0 mM (F). The triphasic rate constants for kAG1 (─), kAG2 (┄), and kAG3 (┈) were calculated from the data in (A).

20 21

Fig. 7. The effects of osmolytes on the reactivation of ECAK.

22

ECAK that was inactivated by 0.04 mM Zn2+ was incubated with different

31

Page 31 of 50

concentrations of glycine (A), proline (B), liquaemin (C), glycerol (D) and sucrose (E)

2

at 20°C for 2 h, and the activity was measured using an assay system in the absence of

3

Zn2+. The final concentration of ECAK was 0.05 μM.

ip t

1

4

Fig. 8. The recovery of the hydrophobic surfaces of ECAK by glycine, proline and

6

liquaemin. The effects of glycine (A), proline (B) and liquaemin (C) treatment were

7

assayed at 20°C for 2 h using the maximum ANS-fluorescence intensity of 0.4 mM

8

Zn2+-treated ECAK. The final concentration of the enzyme was 3.5 μM.

an

us

cr

5

9

Fig. 9. Prevention of ECAK aggregation by glycine, proline and liquaemin.

11

(A) The effect of glycine on ECAK aggregation. The final concentrations of glycine

12

labeled 1 to 7 were 0, 0.2, 0.3, 0.5, 0.6, 1 and 5 mM, respectively. (B) The effect of

13

proline on ECAK aggregation. The final concentrations of proline labeled 1 to 5 were

14

0, 40, 60, 80 and 90 mM, respectively. (C) Effect of liquaemin on ECAK aggregation.

16 17 18 19

d

te

Ac ce p

15

M

10

The final concentrations of liquaemin labeled 1 to 6 were 0, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml, respectively. The final concentrations of Zn2+ and ECAK were 2.0 mM and 20 μM, respectively. The reaction was conducted at 20°C.

Fig. 10. Homology protein model of ECAK.

20

(A) Protein structure model of the homology structure drawn using a cartoon image.

21

(B) Radius of gyration plot. ECAK is the spot in the green filled circle. The red dots

22

represent the Rg values from PDB X-ray structures. The blue line is curve fit to the

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red dots (y = 2.83 x0.34: y=Rg and x=protein size). (C) Distance heat-map plot. The X-

2

and Y-axes show residue numbers. Shorter distances between two residues have

3

darker points.

ip t

1

4

Fig. 11. Predicted ADP binding site of ECAK.

6

(A) (Left figure) homology model with the locations of predicted ADP binding sites

7

(yellow spheres) and pockets (yellow and red spheres). (right figure) Heat-map-like

8

DPSP score. The X-axis shows the pocket numbers found in the ECAK structure. The

9

Y-axis shows the found ligand name. The DPSP scores ranged from -1 (wrong) to 1

10

(right prediction). (B) 3D visualization of ADP entrance interacting residues with Zn2+.

11

The large arrow is considered as the ADP entrance. The ADP binding region is drawn

12

by yellow strands. Three residues (THR304, ARG305, ASP317; red spheres) are

13

interacting with two Zn2+ (blue arrows).

15 16 17 18

us

an

M

d

te

Ac ce p

14

cr

5

Fig. 12. Time profiles of molecular dynamics simulations. (A) RMSDs with (red) and without (green line) Zn2+ with respect to the initial structure. (B) The number of bound Zn2+ analyzed in a time profile. There are 9 ions in total. The used bound condition is a distance below 5 Å from any atoms of the

19

protein. (C) The RMSF plot was calculated to determine the flexibility of the protein.

20

The box symbols above the X-axis indicate the residue positions of bound Zn2+ in the

21

final (red) and initial structures (green box symbols: from the beginning of

22

simulation). Blue box symbols represent the ADP-binding sites predicted from the

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1

DPSP calculation (Fig. 11A).

2

Fig. 13. The final structure of ECAK with the secondary structure scheme.

4

(A) Superimposition of the two final ECAK structure drawn in red (with) and blue

5

(without Zn2+) cartoons. (B) Secondary structures of two final structures (str1: with

6

ions; str2: without ions). Helix/sheet/coil structures are drawn using red/yellow/blue

7

schematic drawings.

us

cr

ip t

3

an

8 9

M

10

Ac ce p

te

d

11

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Fig. 1

1 2

an

us

cr

ip t

A

3

M

4

5 6

Ac ce p

te

d

B

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Fig. 2

1 2

an

us

cr

ip t

A

3

M

4

5 6 7

Ac ce p

te

d

B

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Fig. 3

1 2

an

us

cr

ip t

A

3

M

4

5 6 7 8

Ac ce p

te

d

B

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Fig. 4

1 2

an

us

cr

ip t

A

M

3 4

5

Ac ce p

te

d

B

6 7 8 9

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Fig. 5

1 2

an

us

cr

ip t

A

3

M

4

5 6

Ac ce p

te

d

B

7 8 9 10

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Fig. 6

1 2

B

3

D

5 6

te

E

F

Ac ce p

4

d

M

an

C

us

cr

ip t

A

7 8 9 10

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Fig. 7

1 2

B

us

cr

ip t

A

D

5 6

7 8 9 10 11 12

Ac ce p

E

te

d

M

C

an

3 4

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Fig. 8

1 2

us

cr

ip t

A

an

3 4

5 6

Ac ce p

te

d

M

B

C

7 8

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Fig. 9

1 2

an

us

cr

ip t

A

3

4

Ac ce p

te

d

M

B

C

5 43

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Fig. 10

1

M

an

us

cr

ip t

A

Ac ce p

te

B

d

2

3

44

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an

us

cr

ip t

C

M

1

Ac ce p

te

d

2

45

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Fig. 11

1

M

B

5 6

Ac ce p

te

d

2 3 4

an

us

cr

ip t

A

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Fig. 12

1

A

M

an

us

cr

ip t

2

B

Ac ce p

5

te

4

d

3

6 7 47

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an

us

cr

ip t

C

1

M

2 3

7

te

6

Ac ce p

5

d

4

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Fig. 13

1 2

an

us

cr

ip t

A

4

M

3

B

Ac ce p

te

d

5

6 7

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1 2 3

Highlights for review: Arginine kinase from Exopalaemon carinicauda (ECAK) inactivation mechanisms by Zn2+ ligand binding. Kinetic analysis of Zn2+-induced ESAK aggregation.

5

Several osmolytes roles for preventing ECAK aggregation and recovering

8 9

cr us

7

activity.

Gene cloning of ECAK via RACE and construction of 3D structure by homology prediction.

an

6

ip t

4

Computational molecular dynamics simulation between ECAK and Zn2+.

M

10

Ac ce p

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d

11

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