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Bioactive Constituents, Metabolites, and Functions
Studies on the interaction between angiotensin converting enzyme (ACE) and ACE inhibitory peptide from Saurida elongata Xiongdiao Lan, LiXia Sun, Yaseen Muhammad, Zefen Wang, Haibo Liu, Jianhua Sun, Liqin Zhou, Xuezhen Feng, Dankui Liao, and Shuangfei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04303 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018
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Journal of Agricultural and Food Chemistry
Studies on the interaction between angiotensin converting enzyme (ACE) and ACE inhibitory peptide from Saurida elongata
Xiongdiao Lan†, §, Lixia Sun‡, Yaseen Muhammad‡, II, Zefen Wang‡, Haibo Liu‡, Jianhua Sun‡, Liqin Zhou‡, Xuezhen Feng‡, Dankui Liao*‡, Shuangfei Wang*†
†College
of Light Industry and Food Engineering, Guangxi University, Nanning
530004, China ‡ School
of Chemistry and Chemical Engineering, Guangxi University, Nanning,
Guangxi 530004, China § School
of Chemistry and Chemical Engineering, Guangxi University for
Nationalities, Nanning, Guangxi 530008, China II Institute
*
of Chemical Sciences, University of Peshawar, 25120, KP, Pakistan
Corresponding authors.
Tel: +86 771 327 2702. Fax: +86 771 323 3718 E-mail address:
[email protected];
[email protected] 1
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ABSTRACT
2
Angiotensin converting enzyme (ACE) inhibitory peptides derived from food
3
protein exhibited antihypertensive effects by inhibiting ACE activity. In this work, the
4
interaction between ACE inhibitory peptide GMKCAF (GF-6) and ACE was studied
5
by isothermal titration calorimetry, molecular docking, UV absorption spectroscopy,
6
fluorescence spectroscopy and circular dichroism spectroscopy. Experimental results
7
revealed that the binding of GF-6 to ACE was a spontaneous exothermic process
8
driven by both enthalpy and entropy. The interaction occurred via a static quenching
9
mechanism and involved the alteration of the conformation of ACE. In addition, ITC
10
and molecular docking results indicated binding of GF-6 to ACE via multiple binding
11
sites on the protein surface. This study could be deemed helpful for the better
12
understanding of the inhibitory mechanism of ACE inhibitory peptides.
13 14
KEYWORDS: Angiotensin converting enzyme inhibitory peptides; static quenching
15
interaction; molecular docking; isothermal titration calorimetry; circular dichroism
16
spectroscopy
17 18 19 20 21 22 2
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INTRODUCTION
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Hypertension is a ubiquitously known cardiovascular disease which has become an
25
important global public health issue. 1, 2 Angiotensin-converting enzyme (ACE) is a
26
key enzyme of the renin-angiotensin and kallikrein-kinin system.
27
important role in the regulation of blood pressure and cardiovascular functions.
28
Inhibiting the ACE activity is regarded as a useful treatment against hypertension.
29
Thus, ACE is considered as an appropriate target for the development of
30
antihypertensive drugs.
3, 4
It plays an
31
Since ACE inhibitor was identified in snake venom, many studies have been
32
conducted for synthesizing ACE inhibitors, such as captopril, enalaprilat and lisinopril.
33
Although the ACE inhibitor is the mainstay of hypertension therapy, it can produce
34
substantial side effects such as cough, rash and renal dysfunction. 5-7 And this recently
35
brought a greater attention for new therapeutic agents. Numerous ACE inhibitory
36
peptides derived from food proteins with lesser and milder side effects have been
37
developed as potent alternatives to synthesized and market-available drugs for
38
preventing hypertension.
39
addressed the preparation, purification and amino acid sequence analysis, while the
40
direct information on the interaction of ACE inhibitory peptide and ACE was quite
41
limited.
42
could help us increase and improve our understanding of the influence of ACE
43
inhibitory peptides in vivo and guide the suitable use of these peptides. In this
44
connection, many studies on the interactions between ACE and ACE inhibitory
10-12
8, 9
Earlier studies on ACE inhibitory peptides mainly
Studying the interaction between ACE inhibitory peptides and ACE
3
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45
peptides have been reported. Fu et al.
46
peptides via fluorescence and simulated their interactions usingmolecular docking.
47
Xie et al.
48
according to molecular docking and isothermal titration calorimetry (ITC) assay.
49
These studies have provided information about the binding of ACE inhibitory
50
peptides to ACE, but the mechanism has not been fully explored. Detailed and
51
systematic thermodynamic, spectroscopic and computational studies on the interaction
52
of peptides with ACE have not been reported in literature so far. Keeping in view the
53
reputation earned by spectroscopic, calorimetric and molecular docking techniques in
54
studying the protein-ligand interactions,
55
investigating the interaction between ACE inhibitory peptide and ACE.
56
14
analyzed the inhibition of ACE by patatin
studied the interaction mechanisms of TTW and VHW with ACE
In our previous work,
18
15-17
it is worth exploring their role in
a novel ACE inhibitory peptide GMKCAF (GF-6) with
57
IC50 value of 45.7 μM was isolated from lizard fish (Saurida elongate) protein
58
hydrolysate. In continuation to this, the current study is focused to get insight about
59
the inhibition of GF-6 on ACE via calorimetric, molecular docking and spectroscopic
60
methods. ITC provided the binding constant, binding stoichiometry, enthalpy, entropy
61
and free energy of GF-6-ACE interaction. Structural changes of ACE were analyzed
62
by UV, fluorescence and circular dichroism (CD) spectroscopy. In addition, the mode
63
of binding of peptide with ACE was simulated through molecular docking. This study
64
could be beneficial for understanding the pharmacological and structural changes
65
during the binding of ACE inhibitory peptides with ACE.
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MATERIALS AND METHODS 4
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Materials. ACE was prepared from pig lung according to reported literature.19,20
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Hippuryl-histidyl-leucine (HHL) as a substrate of ACE, was purchased from Sigma
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Chemical Co. (St. Louis, MO, USA). GF-6 (grade ﹥ 98%, IC50= 45.7 μM) was
70
synthesized by GL Biochem (Shanghai) Ltd. (China). All the the other reagents were
71
of analytical purity and used without further purification.
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Inhibition pattern of GF-6. ACE, HHL and GF-6 were dissolved in 0.1 M borate
73
buffered saline (0.3 M NaCl, pH 8.3). A mixture of ACE and GF-6 was incubated in a
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water bath at 37.0 °C for 10 min. 40 μL of HHL in different concentrations (1.16 μM,
75
2.32 μM, 3.49 μM, 4.66 μM, 5.24 μM and 5.82 μM) were added to the mixture. The
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reaction was carried out at 37.0 °C for 5 min and was stopped by adding 150 μL of
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1.0 M HCl. The hippuric acid liberated in the mixture was determined by reverse
78
phase high pressure liquid chromatography (RP-HPLC) with an XDB-C18 (4.6×150
79
mm) column. The inhibition pattern of GF-6 was determined by Lineweaver-Burk
80
plot analysis, a double reciprocal plot of substrate concentration versus velocity
81
according to Michaelis-Menten kinetics.
82
Molecular docking. Although the crystal structure of ACE is not clear yet, human
83
testicular ACE (tACE) (obtained from the Protein Data Bank (1O8A.pdb)) can fully
84
replicate the function of ACE and was used as the macromolecular receptor in
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docking studies. The 3D structure of GF-6 was drawn in ChemDraw 16.0 and saved
86
in .pdb format for docking studies. The Auto Dock Tools transformed the files
87
from .pdb format to .pdbqt format and determined the docking box on the target.
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Molecular docking was performed using the Autodock Vina software.21 Ligand 5
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displaying the lowest binding energy in the binding pocket of protein was chosen as
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the best conformation. PyMOL was used for viewing the diagrams of protein-ligand
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interaction.
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ITC analysis. ITC measurements were performed on a Nano ITC calorimeter (TA,
93
USA). ACE and GF-6 solutions were dissolved in 50 mM HEPES buffer solution (0.3
94
M NaCl, pH 7.0) and properly degassed prior to titration. In a standard experiment,
95
the reference cell of the calorimeter was loaded with HEPES buffer solution (0.3 M
96
NaCl, pH 7.0). After stabilizing the baseline, ACE solution (2.17 μM) in the sample
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cell was titrated with GF-6 solution by 20 successive automatic injections of 2.5 μL
98
each at a stirring speed of 250 rpm. The individual injections were programmed at
99
intervals of 300 s, while similar experiments were performed at 15 °C, 20 °C and
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25 °C. In order to subtract the background dilution heats from the experimental data, a
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control experiment was carried out by injecting GMKCAF solution into the buffer
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alone.
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UV spectroscopic measurements. UV spectra was measured on a UV-2501PC
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Spectrophotometer (Shimadzu, Japan) attached with an externally isothermal water
105
bath to maintain temperature at 15 °C, 20 °C and 25 °C. ACE and GF-6 were
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dissolved in 50 mM HEPES buffer solution (0.3 M NaCl, pH 7.0). A constant
107
concentration of ACE (1.98 μM) was mixed with increasing concentration of GF-6.
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The mixture was incubated at enactment temperature for 10 min. UV absorption
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spectra were recorded in a wavelength range of 200-320 nm, which were corrected
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with respective blank having same concentration of GF-6 in buffer without ACE. 6
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Fluorescence spectroscopic measurements. Fluorescence measurements were
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performed on a LS-55 Fluorescence spectrophotometer (PE, USA). GF-6 solution was
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prepared with 50 mM HEPES buffer (0.3 M NaCl, pH 7.0) at different concentration
114
and then incubated with 0.0718 μM ACE for 10 min. The emission spectra were
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recorded in wavelength range of 300-500 nm at an excitation wavelength of 285 nm.
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The excitation and emission slit widths were set at 5 nm at variable temperatures of
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15 °C, 20 °C and 25 °C using an isothermal water bath.
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CD spectroscopic measurements. CD spectral data were obtained from a
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MOS 450 CD spectrometer (Biologi, French) in rectangular quartz cuvettes of 1 mm
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path length. GF-6 solution was prepared with 50 mM HEPES buffer at different
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concentration followed by incubation with 1.12 μM ACE for 10 min. Spectra were
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recorded from 190 to 260 nm with 100 nm·min-1 scan speed and a response time of 1
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s. The background spectrum of GF-6 solution was subtracted from that of GF-6-ACE
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complex. The secondary structures of the samples were estimated from the CD spectra
125
using SELCON 3 method in Dicroprot 2000 software.
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Statistical analysis. All measurements performed in triplicate and analysis of data
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variance was performed using statistical analysis system (SAS 9.1). Linear correlation
128
coefficient was calculated using Origin 8.0. Values reported as significantly different
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have P values ﹤0.05.
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RESULTS AND DISCUSSION
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Inhibitory pattern of GF-6 on ACE. The inhibition kinetics of GF-6 was studied
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at different substrate concentrations, as well as varying the inhibitor level in the assay 7
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and the Lineweaver-Buck plots of the reactions are shown in Fig. 1. The lines
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intersect on 1/[s] axis indicates that GF-6 can prevent product formation though
135
binding with ACE at the non-active site as a non-competitive inhibitor.
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peptides derived from food proteins such as VHW, TTW, 14 TPTQQS, 22 RYDF and
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GNGSGYVSR
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synthetic drugs (captopril, enalapril and lisinopril) are competitive inhibitors
23
22
Some
have also been found to act in non-competitive pattern, while
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Molecular docking. Molecular docking of GF-6 with human tACE was carried out
140
using AutoDock Vina to predict the ligand-receptor interaction mechanism. The pose
141
with the lowest binding energy was recognized as the best conformation for further
142
analysis.
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According to the docking results, the most stable structure of GF-6 in the
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hydrophobic cavity of tACE had a binding affinity of -7.0 kcal·mol-1, as shown in Fig.
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2a. The hydrogen bond (H-bond) could play an important role in GF-6 binding with
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tACE (Fig. 2b). The main active site of ACE is composed of three pockets i.e. S1
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pocket (Gln281, His353, Lys511, His513), S2 pocket (Ala354, Glu384, and Tyr523)
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and S1’ pocket (Glu162), which are considered as the probable contacting sites of
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competitive inhibitors with tACE. 17, 18 No interactions of GF-6 with Zn2+ is observed
150
(Fig. 2b). On the other hand, GF-6 is attached to tACE primarily by the residues
151
(Ser-480, Ser-481, Ala-320 and Arg-486), which were not loaded in the three active
152
pockets. Protein-ligand interactions show four H-bonds with tACE. The Lys of
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peptide makes the greatest contribution to the stability of the peptide-ACE complex,
154
owing to its two H-bonds with Ser-480 and Ser-481 (2.4 Å and 2.2 Å). The Cys 8
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formed one H-bond with the residues Arg-486 (2.3 Å) and Ala bound to Ala-320
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having a bond distance of 2.5 Å. These interactions kept the peptide away from the
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active site. However, previous structure-activity correlation studies have shown that
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C-terminal tripeptide of peptides plays a predominant role in binding to the active site
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of the enzyme, where position C-1 is the most relevant to the inhibitory activity. This
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disagreement may be due to the fact that these studies are based on a competitive
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inhibition mechanism in which peptides interact with the active site of ACE, while
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GF-6 (in the current study) is a non-competitive inhibitor.
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ITC assays of binding of GF-6 to ACE. The thermodynamic profiles of
164
GF-6-ACE interactions obtained by ITC are shown in Fig. 3. The upper panels show
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the raw ITC profiles for the complexation of GF-6 with ACE. Each of the heat burst
166
peaks corresponds to a single injection of GF-6 into the ACE solution, while the lower
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panels correspond to the corrected heats per mole of injection. The association
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constant (Ka), binding stoichiometry (n), and enthalpy change (∆H) were obtained
169
after fitting of the integrated heats. The Gibbs free change (∆G) and change in entropy
170
(-T∆S) were calculated using Eq. (1) and Eq. (2) respectively. 24 The thermodynamic
171
parameters accompanying the binding of GMKCAF to ACE are summarized in
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Table 1.
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∆G = - RT ln(Ka)
(1)
∆G = ∆ H - T ∆ S
(2)
Where R is the general gas constant and T is the absolute temperature (K).
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Table 1 suggests a spontaneous exothermic nature of the binding process of GF-6 to
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ACE. The values of ΔG, ΔH and -T∆S decipher valuable information about the
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different binding forces involved. 25 ,26 These results further suggested that the binding
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process is concurrently driven by enthalpy and entropy. The negligible contribution of
178
entropy at lower temperature implies the major role of H-bonds and electrostatic
179
interactions in the process. The binding has more favorable entropy contribution at
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high temperature, suggesting that GF-6 comes in proximity to ACE through
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hydrophobic force.
182
The interaction between ligand and receptor contains two types of binding: specific
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and nonspecific. “Specific binding” represents the “lock-key” interaction with strong
184
affinity, while “non-specific binding” refers to the binding of non-physiological origin
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with weak affinity. 27,28 The values of association constant Ka between GF-6 and ACE
186
were 4.67×105, 1.63×104 and 9.78×103 M-1 at 15 °C, 20 °C and 25 °C, respectively,
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while those reported for captopril, enalaprilat and lisinopril are 7.5×107, 1.9×108, and
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6.2×108 M-1, respectively. 29 These studies showed that all the three inhibitors bound
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with ACE at the active site via “specific binding”, while GF-6 interacted with ACE
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via “nonspecific binding”.
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Binding stoichiometry (n) values show the possibility of multiple binding sites on
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ACE for GF-6, which increased with increasing temperature. It may be due to the
193
variation in ACE structure at variable temperature. Residues (exposed or buried) can
194
enhance ACE interaction with GF-6. As seen from Fig. 3, saturations of binding sites
195
occurred with molar ratio of approximately 67:1, 294:1 and 392:1 at 15 °C, 20 °C and 10
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25 °C, respectively. On the contrary, the saturated bindings of captopril, enalaprilat
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and lisinopril with ACE have been reported at a molar ratio of 1:1.30 The sequence of
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somatic ACE contains two potential active sites. The excessively large values of “n”
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confirm the non-active nature of these binding sites. The interaction of tannins with
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proteins (in tannin-protein binding) is primarily a surface phenomenon with higher
201
number of binding sites as supported by stoichiometric studies.31-33 From ITC results,
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large number of GF-6 binding sites indicate a physiologically relevant binding and
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involves surface adsorption mechanism. A possible explanation is that ACE structure
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is flexible to transform to other conformations, and hence can provide more surface
205
binding sites for non-competitive peptide. However, our molecular docking
206
simulation suggested that GF-6 attached to the hydrophobic cavity of ACE. It may be
207
due to the fact that docking was performed around the active site (x, 43.642; y, 38.406
208
and z, 46.282) with the size of docking box as: 60 Å×60 Å×60 Å, and possible surface
209
binding was not considered. To address this issue, a re-docking was performed by
210
selecting the search box as large as possible to cover the entire ACE surface and the
211
results are provided in Fig. 4.
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Re-docking results showed that GF-6 interacted with ACE surface with a binding
213
affinity of -7.5 kcal·mol-1 whereas around active site it was -7.0 kcal·mol-1. The
214
binding tends to occur on the ACE surface rather than hydrophobic cavity, which is
215
confirmed from ITC assay. Fig. 4b suggests many grooves on the surface of ACE
216
which equip GF-6 with large number of binding sites. In GF-6-ACE complex, peptide
217
lies on the surface of ACE and makes close contacts with enzyme through H-bond 11
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formation with Thr-135, and Thr-266, Asn-249, Leu-339, and Glu-340. Where, Lys of
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peptide forms two H-bonds with Thr-135 and Thr-266, (2.1 Å and 2.2 Å); Cys forms
220
three H-bonds with Asn-249, Leu-339, and Glu-340 (2.1 Å and 2.2 Å); and Ala forms
221
one H-bond with Asn-249. These results suggested that Cys makes the greatest
222
contribution to the stability of peptide bound with ACE.
223
Recently, numerous ACE inhibitory peptides have been identified from protein
224
hydrolysates, most of which are non-competitive inhibitor (bound to non-active sites)
225
with much higher IC50 values than those of the competitive inhibitors. Previously, the
226
inhibitory mechanisms of several ACE inhibitory peptides (competitive and
227
non-competitive) were assumed to bind with ACE in the hydrophobic cavity at a ratio
228
of 1:1.14,
229
However, the exact location of non-active sites is unclear and hence these models may
230
not be applicable. In order to predict and investigate the binding of these inhibitors,
231
docking simulation should be carried out in the whole protein molecular structure.
34, 35
This binding model may explain the competitive inhibition behavior.
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Effects of GF-6 binding on the UV absorption spectra of ACE. The absorption
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spectra of ACE at different temperature shown in Fig. 5a suggest two absorption
234
peaks at 226 nm and 280 nm. The strong absorption peak at 226 nm is characteristic
235
of the peptide backbone, while a weaker absorption peak around 280 nm is typical of
236
aromatic amino acids.36 The intensity of the peak at 226 nm dramatically changed due
237
to red shifts with increasing temperature from 15 °C to 20 °C, indicating the
238
disturbances to the unfolding of ACE skeleton. Further increasing temperature to
239
25 °C shifts the peak at 226 nm to shorter wavelength, suggesting the folding of ACE 12
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skeleton. The intensity of the peak at 280 nm increases obviously with the increasing
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temperature indicating the exposure of buried aromatic amino acids residues.
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To further investigate the binding of GF-6 to ACE, spectral experiments were
243
performed according to ITC results (Table 2). The UV absorption spectra of
244
GF-6-ACE complex were obtained at constant ACE concentration while varying
245
GF-6 concentration. Fig. 5b-d suggest changes in absorption spectra of ACE with the
246
addition of GF-6 indicating binding interactions between the two. The intensity of
247
peak at 280 nm is slightly affected with increase in GF-6 concentration, while that of
248
226 nm peak shifts to longer wavelength (redshift). These results suggested that the
249
binding interaction changed the hydrophobicity around the amide bonds and reflected
250
the unfolding of peptide backbone. Meanwhile, the environment around aromatic
251
amino acids was also affected. The different binding model might result in different
252
changes of ACE under diverse conditions. Moreover, combination with a large
253
number of peptides could lead to the denaturation of ACE, which provided ACE with
254
a flexible pattern and hence able to change in the presence of excessive GF-6.
255
Effect of GF-6 binding on fluorescence of ACE. Intrinsic fluorescence of proteins
256
is mainly due to two aromatic amino acid residues, tryptophan (Trp) and tyrosine (Tyr)
257
when excited at 280 nm. The fluorescence spectra of ACE at different temperature
258
shown in Fig. 6a suggest a λmax of 341 nm at 15 °C, which shifted to 338 nm and 339
259
nm at 20 °C and 25 °C respectively. It has been reported that residues inside the
260
protein show a shorter λmax than those on or near the surface of protein. The decrease
261
in λmax may be due to the movements of Trp and Tyr residues to more hydrophobic 13
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environments. The variation in λmax demonstrated a significant effect of temperature
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on the ACE structure. 37-39
264
The fluorescence spectra of ACE having various concentration of GF-6 at different
265
temperatures are shown in Fig. 6b-d. The fluorescence intensity of ACE decreases
266
with the addition of GF-6, which is attributed to the fluorescence quenching process
267
by GF-6. In order to elucidate the quenching mechanism, fluorescence data were
268
analyzed according to the Stern-Volmer Equation as Eq. (3):
269
F0/F=1+Kq∙τ0∙[Q]=1+KSV∙[Q]
(3)
270
Where, F0 and F are fluorescence intensities of ACE in the absence and presence of
271
quencher, respectively. Kq is the quenching rate constant of biomolecular reaction, τo
272
is the average fluorescence lifetime of the biomolecule in the absence of quenchers,
273
which is of the order of 10-8 s. KSV is the Stern-Volmer quenching constant and and
274
[Q] is the concentration of the quencher.
275
The Stern-Volmer plot of Fo/F versus [Q] at 25 °C is presented in Fig. 6e. The plot
276
is linear before ACE saturation by GF-6, while the quenching is not regular after
277
saturation. Thus, the fluorescence data before saturation at 15 °C, 20 °C, and 25 °C
278
were fitted using the Stern-Volmer Equation and provided in Fig. 6f. Good linear
279
relationships between F0/F and [Q] were observed, and the values of Ksv decreased
280
with increasing temperature (Table 3). In addition, the values of Kq are greater than
281
the maximum value for dynamic quenching (2 × 1010 M-1·s-1). These results suggested
282
that the fluorescence quenching of ACE by GF-6 was a static process and was
283
initiated by the formation of a GF-6-ACE complex, not from dynamic collision. 40 14
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Conformation changes of ACE upon interaction with GF-6. Proteins with
285
different structures provide different CD band positions and intensities.41,42 In Fig. 7,
286
the CD spectra of ACE varied significantly with increasing concentrations of GF-6.
287
For the native ACE, the contents of the secondary structural elements are of about 7.9%
288
α-helices, 23.3% β-sheets, 10.3% β-turns, 9.1% P2 and 50.0% random coils. The low
289
content of α-helices may be the major factor providing a flexible structure of ACE to
290
contact with greater number of GF-6, while the proportion of α-helices varies greatly
291
from that found in the crystal structure of tACE. It may be attributed to the fact that
292
ACE crystals were grown at 16 °C under pH 4.7, 3 while certain structure of protein
293
survives only under specific pH and temperature. As seen from Table 4, the α-helical
294
and β-sheet content of ACE decreased upon the addition of GF-6 to the ratio of 113:1,
295
revealing that GF-6 associates with the amino acid residues of ACE and destroys their
296
H-bonding networks. Upon saturation of ACE and GF-6, the contents of α-helix and
297
β-sheet of ACE increase to 13.2% and 30.3%, respectively. This may be attributed to
298
the larger number of GF-6 binding on ACE according to the surface adsorption
299
mechanism. Compare with the saturation state, variations in the conformation of ACE
300
structures are observed even at excessive amount of GF-6 than ACE. This is in
301
accordance with UV absorption spectra and fluorescence spectra results (Fig. 5b-d).
302
In summary, this study though ITC, molecular docking, UV spectroscopy,
303
fluorescence spectroscopy and CD spectroscopy, provided important quantitative data
304
for the interaction of GF-6 with ACE. Experimental results revealed that the binding
305
between GF-6 and ACE was based on complexation. GF-6 combined with ACE at 15
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non-active site as non-competitive inhibitor and effectively quenched ACE via static
307
quenching mechanism. ITC and spectroscopic studies revealed that the main driven
308
force changed from H-bonds to hydrophobic force with increasing temperature, which
309
was due to the flexible structure of ACE. For the GF-6-ACE complex, surface
310
adsorption mechanism explained the inhibition of the non-competitive peptides with
311
higher number of binding sites, which was supported by the molecular docking results.
312
This study provides important insights into the binding of GF-6 with ACE and can
313
help better understand the mechanism and mode of action of ACE inhibitory peptides.
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A.G.; Asiwu, R.Y. Adherence to lifestyle modification among hypertensive clients: A
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Funding
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Journal of Agricultural and Food Chemistry
This research was supported by the National Natural Science Foundation of China
443
(51372043),
Guangxi
Natural
Science
Foundation
444
2017GXNSFBA198215, 2017GXNSFAA198289)
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(2017GXNSFDA198052,
Journal of Agricultural and Food Chemistry
Figure captions: Figure 1: The Lineweave-Burk plots of the reactions of ACE in the presence of GF-6. Figure 2: The docking simulation of GF-6 binding to tACE. (a)The docking simulation of GF-6 (green) binding to tACE (shown as cartoon). A zinc ion (red) was present in the active site of tACE. (b)The interaction between GF-6 (shown as sticks) and the residues of tACE (shown as lines) is shown.
Figure 3 The heat flow of binding GF-6 to ACE (2.17 μM). (a) at 15°C,concentration of GF-6 is 1.28 mM; (a) at 20°C, concentration of GF-6 is 5.12 mM; (a) at 25°C, concentration of GF-6 is 5.12 mM Figure 4: The second molecular docking simulation of GF-6 binding to tACE. (a)The docking simulation of GF-6 (green) binding to tACE (shown as cartoon). A zinc ion (red) was present in the active site of tACE. (b) The binding of the GF-6 (green) on the surface of the ACE (orange). (c)The interaction between GF-6 (shown as sticks) and the residues of tACE (shown as lines) is shown. Red dash indicates H bonding. Figure 5: UV absorption spectra of ACE in the absence and presence of GF-6 at different concentrations. Concentration of ACE is 1.98 μM; (a) without GF-6; (b) 15°C; (c) 20°C; (d)25°C. Figure 6: Fluorescence emission spectra of ACE (0.0718 μM). (a) without GF-6; (b) 15°C; (c) 20°C; (d)25°C; (e)emission quenching curves of ACE (0.0718 μM) at 25°C; (f) emission quenching curves of ACE (0.0718 μM) at 25°C at all studied temperature Figure 7: CD spectra of ACE in the absence and presence of GF-6 at 25°C. CACE = 1.12 μM. 24
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Table 1 Thermodynamics parameters of binding between GF-6 and ACE T(°C)
n
Ka(1/M)
∆H(KJ·mol-1)
∆G(KJ·mol-1)
-T∆S(kJ·mol-1)
15
26.2
4.67×105
-31.20
-31.27
-0.07
20
103.7
1.63×104
-2.28
-23.64
-21.36
136.2
9.78×103
-3.52
-22.78
-19.26
25
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Table 2 Spectroscopic experimental conditions T(°C)
GF-6:ACE
Titration number of ITC
15
0:1
-
28:1
3
67:1
7
119:1
12
212:1
20
0:1
-
113:1
3
294:1
7
611:1
15
849:1
20
0:1
-
113:1
3
392:1
10
611:1
15
849:1
20
Saturation
20
Saturation
25
Saturation
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Table 3 Stern-Volmer quenching constants (KSV and Kq) due to binding between GF-6 and ACE
T (°C) 25 20 15
104
Ksv L/mol
1012
0.94 1.51 5.37
Kq L/mol·S 0.94 1.51 5.37
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Table 4 Secondary structures of ACE affected by GF-6 at 25 °C GMKCAF:ACE
α-helices % (±0.1)
β-sheetss % (±0.2)
β-turns % (±0.1)
P2 % (±0.1)
Random coils % (±0.3)
0:1
7.9
23.3
10.3
9.0
50.0
113:1
5.5
22.8
10.7
9.0
51.0
292:1
13.2
30.3
9.1
9.7
28.0
611:1
11.9
28.6
8.2
9.3
29.0
848:1
12.1
33.6
9.3
10.5
32.0
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Fig. 1
control 30.4M GF-6 60.8M GF-6
1000
-1
1/v(M min)
800
-2
-1
600 400 200 0 -200
0
1
2
3
4
5
-1
1/[S](mM )
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Fig. 2
a
b
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Fig. 3
a
b
c
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Fig. 4
a
b
c
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Fig. 5
a
1.2
b
15C 20C 25C
1.0
GF-6ACE=01 GF-6ACE=281 GF-6ACE=671 GF-6ACE=1191 GF-6ACE=2121
1.2 1.0 0.8 0.6
0.8
0.4 0.2
A
A
0.6 0.4
0.0 -0.2 -0.4
0.2
-0.6 -0.8
0.0
-1.0
220
240
260
280
300
220
320
240
260
c
1.2
0.8 0.6
300
320
1.2 GF-6ACE=01 GF-6ACE=1131 GF-6ACE=3921 GF-6ACE=6111 GF-6ACE=8491
1.0 0.8 0.6
0.4
0.4
0.2
0.2 A
A
d
GF-6ACE=01 GF-6ACE=1131 GF-6ACE=2941 GF-6ACE=6111 GF-6ACE=8491
1.0
280 nm
nm
0.0
0.0
-0.2
-0.2
-0.4
-0.4
-0.6
-0.6
-0.8
-0.8 -1.0
-1.0 220
240
260
280
300
220
320
240
260 nm
nm
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300
320
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Fig. 6
a
300 250
Flourescence Intensity
b
15C 20C 25C
300 GF-6ACE=01 GF-6ACE=281 GF-6ACE=671 GF-6ACE=1191 GF-6ACE=2121
250
200
200
150
F
150
100
100
50
50 0 300
0 300
320
340
360
380
400
420
440
460
480
500
320
340
360
380
400
c
d
300 GF-6ACE=01 GF-6ACE=1131 GF-6ACE=2941 GF-6ACE=6111 GF-6ACE=8491
250 200 F
100
50
50
360
380
400
420
440
460
480
0 300
500
320
340
360
380
400
f
1.20
1.16
1.15
1.12
1.10
1.04
1.00
1.00 10
15
20
440
460
480
1.08
1.05
5
420
25
30
35
15 C 20 C 25 C
1.20
F/F0
F/F0
1.25
0
500
nm
nm
e
480
150
100
340
460
GF-6ACE=01 GF-6ACE=1131 GF-6ACE=3921 GF-6ACE=6111 GF-6ACE=8491
200 F
320
440
300 250
150
0 300
420
nm
Wavelength(nm)
-2
[Q]
0
2
4
6
8 [Q]
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12
14
16
18
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Fig. 7
14000
GF-6:ACE=0:1 GF-6:ACE=113:1 GF-6:ACE=392:1 GF-6:ACE=611:1 GF-6:ACE=849:1
12000 10000
Ellipticities
8000 6000 4000 2000 0 -2000 -4000 -6000 -8000 190
200
210
220
230
240
250
260
Wavelength(nm)
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Table of Contents Graphic (TOC)
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