Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism
Low molecular weight natural mediators, 2'-5'-linked oligoadenylates, play an important role in interferon-based antiviral mechanism; participate in growth, apoptosis and other important cellular processes. The aim of current study was to find the evidence for the cell interaction with hum...
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Інститут молекулярної біології і генетики НАН України
2014
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nasplib_isofts_kiev_ua-123456789-1544342025-02-09T22:48:19Z Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism Вивчення впливу дефосфорильованих 2'-5' олігоаденілатів на конформацію білка apo-S100A1 методами гетероядерного ЯМР та кругового дихроїзму Изучение влияния дефосфорилированных 2'-5' олигоаденилатов на конформацию белка apo-S100A1 методами гетероядерного ЯМР и кругового дихроизма Skorobogatov, O.Yu. Lozhko, D.N. Zhukov, I.Yu. Kozlov, O.V. Tkachuk, Z.Yu. Structure and Function of Biopolymers Low molecular weight natural mediators, 2'-5'-linked oligoadenylates, play an important role in interferon-based antiviral mechanism; participate in growth, apoptosis and other important cellular processes. The aim of current study was to find the evidence for the cell interaction with human apo-S100A1 using the methods of circular dichroism (CD) and heteronuclear NMR spectroscopy. Taking into account their concentration within living cells, the 2'-5'A3 oligoadenylates may act as additional biologically active substrates, capable of regulating the S100A1 protein functioning in vivo. The obtained results demonstrated the occurrence of the secondary structure changes in human S100A1 protein upon the interaction with 2'-5'-linked oligoadenylates as well as indicated specific residues involved in this process. Our study points to the 2'-5'-linked oligoadenylates as possible additional elements of the complex system of fine regulation of the Ca2+-signal transduction pathway in human cells. Низькомолекулярні медіатори природного походження – 2'-5' олігоаденілати – відіграють важливу роль в антивірусному механізмі, пов’язаному з інтерфероном, вони причетні до росту клітин, апоптозу та інших важливих процесів, що протікають у клітині. Мета даного дослідження полягала в пошуку доказів мож- ливості взаємодії 2'-5' олігоаденілатів з апо-формою білка S100A1 людини. Методи. Використано методи ЯМР і КД. Результати. Зважаючи на значну концентрацію 2'-5' олігоаденілатів всередині живої клітини, можна припустити, що вони слугують додатковими біологічно активними субстратами і здатні регулювати функціонування білка S100A1 in vivo. Отримані дані вказують на те, що внаслідок взаємодії між 2'-5' олігоаденілатами та S100A1 відбуваються перебудови у вторинній структурі останнього. Крім того, вдалося визначити, які саме амінокислотні залишки беруть учать у цій взаємодії. Висновки. Ймовірно, 2'-5' олігоаденілати є додатковими елементами складної системи регуляції процесів, опосередкованих іонами Са2+. Низкомолекулярные медиаторы природного происхождения – 2'-5' олигоаденилаты – играют важную роль в антивирусном механизме, связанном с интерфероном, они причастны к росту клеток, апоптозу и другим важным процессам, происходящим в клетке. Цель данного исследования состояла в поиске доказательств возможности взаимодействия 2'-5' олигоаденилатов с апо-формой белка S100A1 человека. Методы. Использованы методы ЯМР и КД. Результаты. Учитывая концентрацию 2'-5' олигоаденилатов внутри живой клетки, можно предположить, что они служат дополнительными биологически активными соединениями, способными регулировать функционирование белка S100A1 in vivo. Полученные данные указывают на то, что в итоге взаимодействия между 2'-5' олигоаденилатами и белком S100A1 происходят изменения вторичной структуры последнего. Кроме того, удалось определить аминокислотные остатки, непосредственно участвующие в этом взаимодействии. Выводы. Вероятно, что 2'-5' олигоаденилаты являются дополнительными элементами сложной системы, регулирующей процессы, опосредованные ионами Са2+. 2014 Article Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism / O.Yu. Skorobogatov, D.N. Lozhko, I.Yu. Zhukov, O.V. Kozlov, Z.Yu. Tkachuk // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 279-285. — Бібліогр.: 35 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008A1 https://nasplib.isofts.kiev.ua/handle/123456789/154434 577.32 : 577.112 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Skorobogatov, O.Yu. Lozhko, D.N. Zhukov, I.Yu. Kozlov, O.V. Tkachuk, Z.Yu. Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism Вiopolymers and Cell |
| description |
Low molecular weight natural mediators, 2'-5'-linked oligoadenylates, play an important role in interferon-based antiviral mechanism; participate in growth, apoptosis and other important cellular processes. The aim of current study was to find the evidence for the cell interaction with human apo-S100A1 using the methods of circular dichroism (CD) and heteronuclear NMR spectroscopy. Taking into account their concentration within living cells, the 2'-5'A3 oligoadenylates may act as additional biologically active substrates, capable of regulating the S100A1 protein functioning in vivo. The obtained results demonstrated the occurrence of the secondary structure changes in human S100A1 protein upon the interaction with 2'-5'-linked oligoadenylates as well as indicated specific residues involved in this process. Our study points to the 2'-5'-linked oligoadenylates as possible additional elements of the complex system of fine regulation of the Ca2+-signal transduction pathway in human cells. |
| format |
Article |
| author |
Skorobogatov, O.Yu. Lozhko, D.N. Zhukov, I.Yu. Kozlov, O.V. Tkachuk, Z.Yu. |
| author_facet |
Skorobogatov, O.Yu. Lozhko, D.N. Zhukov, I.Yu. Kozlov, O.V. Tkachuk, Z.Yu. |
| author_sort |
Skorobogatov, O.Yu. |
| title |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism |
| title_short |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism |
| title_full |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism |
| title_fullStr |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism |
| title_full_unstemmed |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism |
| title_sort |
study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-s100a1 protein conformation by heteronuclear nmr and circular dichroism |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2014 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154434 |
| citation_txt |
Study of dephosphorylated 2'-5'-linked oligoadenylates impact on apo-S100A1 protein conformation by heteronuclear NMR and circular dichroism / O.Yu. Skorobogatov, D.N. Lozhko, I.Yu. Zhukov, O.V. Kozlov, Z.Yu. Tkachuk // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 279-285. — Бібліогр.: 35 назв. — англ. |
| series |
Вiopolymers and Cell |
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STRUCTURE AND FUNCTION OF BIOPOLYMERS
UDC 577.32 : 577.112
Study of dephosphorylated 2'-5'-linked oligoadenylates
impact on apo-S100A1 protein conformation
by heteronuclear NMR and circular dichroism
O. Yu. Skorobogatov1, D. N. Lozhko1, I. Yu. Zhukov2, 3, O. V. Kozlov1, Z. Yu. Tkachuk1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2Institute of Biochemistry and Biophysics, Polish Academy of Sciences
5a, Pawinskiego Str., Warsaw, Poland, 02-106
3NanoBioMedical Centre, Adam Mickiewicz University
85, Umultowska Str., Poznan, Poland, 61-614
skorobogatov.alx@gmail.com
Low molecular weight natural mediators, 2'-5'-linked oligoadenylates, play an important role in interferon-ba-
sed antiviral mechanism; participate in growth, apoptosis and other important cellular processes. Taking into
account their concentration within living cells, the 2'-5'A3 oligoadenylates may act as additional biologically acti-
ve substrates, capable of regulating the S100A1 protein functioning in vivo. Aim. Find the evidence for the interac-
tion of human apo-S100A1 with 2'-5'-linked oligoadenylates. Methods. Using the circular dichroism (CD) and
heteronuclear NMR spectroscopy. Results. The obtained results demonstrated the occurrence of the secondary
structure changes in human S100A1 protein upon the interaction with 2'-5'-linked oligoadenylates as well as indi-
cated specific residues involved in this process. Conclusions. Our study points to the 2'-5'-linked oligoadenylates
as possible additional elements of the complex system of fine regulation of the Ca2+-signal transduction pathway in
human cells.
Keywords: 2'-5'-linked oligoadenylates, S100A1.
Introduction. Triphosphorylated 2'-5'-linked oligoade-
nylates, which can be defined by the general formula
ppp(2'- 5')An, are being synthesized in the cell through
interferon-induced enzyme – 2'-5'-oligoadenylate syn-
thase (OAS). These compounds play a key role in the
antiviral innate immunity mechanism, participate in the
cell differentiation and apoptosis processes, at the same
time being involved in diabetes and atherosclerosis pa-
thogenesis. It was demonstrated, that they can be used
as effective therapeutic compounds for oncology and
hematology treatment [1, 2]. Their biological activity is
mainly related to the ribonuclease L (RNase L) activa-
tion: only phosphorylated 2'-5'-linked oligoadenylates
are capable of activating the enzyme due to the presen-
ce of phosphate groups though. dephosphorylated, or
so-called «core» 2'-5'-linked oligoadenylates, do not bind
to RNase L thus leaving the enzyme inactive [1–3].
The long-term research of 2'-5'-linked oligoadeny-
lates is based on the «interferon hypothesis» of biolo-
gical activity [1–4]. It assumes that the antiviral acti-
vity of these compounds is based on the formation of
phosphorylated 2'-5'-linked oligoadenylates and further
activation of RNase L, which specifically cleaves viral
mRNAs, therefore rendering their antiviral properties.
Nevertheless, phosphorylated oligoadenylates possess
a low stability within the cell: they are cleaved by phos-
phorylases, thus forming «core» oligoadenylates, which
display a set of other activities that have nothing to do
with the interferon–RNase L system. In particular, 2'-
5'-linked triadenylates (2'-5'A3) and their analogues
possess cardio protector potential and may act as graft
versus host disease (GVHD) inhibitors [5]. It is worth
279
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 4. P. 279–285 doi: http://dx.doi.org/10.7124/bc.0008A1
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
mentioning, that these compounds are also capable of
executing the inhibiting effect on smooth muscle con-
traction [6].
The biological action of «core» 2'-5'A3 is not fully
understood yet. Up to now, all target proteins are un-
known. Our previous experiments identified a set of
protein targets, with which the «core» 2'-5'A3 and its
analogues interact: strong binding to albumin and inter-
feron and weak binding to immunoglobulin were de-
monstrated [7]. In order to investigate the ability of
«core» 2'-5'A3 to interact with �-interferon – the key
protein of 2'-5'OAS/RNAase L system, responsible for
antiviral cell defense – MALDI-TOF mass spectromet-
ry was applied. It was shown, that naturally occurring
2'-5'A3 and its epoxy-modified analogue (2'-5'A3-epo)
bind to a-interferon, so 3'-5'-linked triadenylate does.
Simultaneous binding of up to five ligand molecules
was demonstrated [8].
It is a matter of common knowledge that Ca2+ signal
transduction is an essential mechanism, ensuring various
cell processes regulation. Multifunctional Ca2+-trans-
ducing protein Calmodulin (CaM) is one of the key pro-
teins involved in it. As it has recently been shown in our
group, native 2'-5'A3 binding to human CaM caused an
alteration of its functional profile [9]. The experimental
data revealed considerable (2–3 times) increase of its
Ca2+ affinity upon interaction with «core« 2'-5'A3, its
epoxy and 3'-cordycepin modified analogues.
Using these data as a background, we decided to
perform a study, focused on another Ca2+-binding pro-
tein, human S100A1. It is the protein with known acti-
vity during neurological disorders and several types of
cancer; its expression level serves as one of the key in-
dicators for heart failure [10, 11]. As shown recently,
the S100A1 interacts with ryanodine receptor (RyR) in
heart (RyR1) and skeletal (RyR2) muscles [12]. The
S100A1 shares with CaM exactly the same binding site
to interact with RyR receptors [13]. The S100A1, an im-
portant regulator of calcium cycling, revealed the mo-
dulated activity of RyR in calcium-dependent way. The
model activation of calcium release by RyR receptor in
both hearts and skeletal muscles included knock-out
RyR inhibitor (CaM).
Earlier, we have shown that dephosphorylated 2'-
5'A3 and its chemically modified analogues possess si-
milar activity towards stimulating the Ca2+-currents in-
activation kine tics in HVA cell cultures [14]. Other ex-
perimental results, obtained by us, demonstrate that 2'-
5'A3 stimulates the RyR mediated Ca2+ release from the
sarcoplasmatic reticulum in the smooth muscle cells [6].
Current report demonstrates the recently obtained
experimental data, proving the possibility of interac-
tion between the «core» 2'-5'A3 and human S100A1 pro-
tein in apo-(Ca2+ free) form.
Materials and methods. Synthesis of 2'-5'-linked
oligoadenylates. Natural 2'-5'A3 and its epoxy modi-
fied analogue (2'-5'A3-epoxy) were synthesized from a
solution modified by phosphotriether method [15]
(Fig. 1).
Synthesis and purification of the recombinant hu-
man apo-S100A1. The synthetic gene encoding human
S100A1 was cloned into pET-30a + plasmid and ex-
280
SKOROBOGATOV O. Yu. ET AL.
2-5-A3 2-5-epoxy-A3
O
N
N N
N
NH
2
OH OH
O
N
N N
N
NH
2
OH O
P
O
O
O
O
N
N N
N
NH
2
OH
OH
O
P
O
O
O
O
N
N N
N
NH
2O
N
N N
N
NH
2
OH O
P
O
O
O
O
N
N N
N
NH
2
OH
OH
O
P
O
O
O
O
-
- -
-
Fig. 1. Chemical formula of na-
tive 2'-5'A
3
(left) and its epoxy de-
rivative (right). The oxygen atom,
involved in epoxy-group forma-
tion, is high-lighted in blue
pressed in Escherichia coli utilizing the T7 expression
system. Bacterial cells were grown at 37 °C in LB me-
dium. Expression was induced by addition of 0.4 mM
IPTG at OD600 = 0.8. Bacterial culture was grown for 2 h
afterward. Human S100A1 was isolated using the clas-
sical method of ammonium sulfate precipitation [16,
17] followed by the purification procedure using rever-
sed-phase HPLC on a semi preparative Vydac C18 co-
lumn [18]. The final product was identified by the elect-
rospray ionization mass spectrometry using a Macro-
mass Q-Tof spectrometer. The concentration in solu-
tion was estimated from its absorbance at 280 nm. For
the synthesis of the recombinant 15N-labeled human
S100A1 protein, the E. coli cells were grown in M9 me-
dia containing (15NH4)2SO4 as the sole nitrogen source
and unlabeled glucose as the sole carbon source.
CD Spectroscopy. The far-UV CD spectra were re-
corded over 260–200 nm range on Jasco J-815 CD
spectropolarimeter at 298 K in a 2 mm light path length
cuvette. For the measurements, 8 �M human Ca2+-free
form of S100A1 protein (apo-S100A1) was dissolved in
a buffer that contained 5 mM Tris-HCl and 100 mM
NaCl (pH 7.5). The concentration of human S100A1 in
solution was controlled spectrophotometrically by mea-
suring its UV absorbance at 280 nm using extinction co-
efficient 10200 (mol–1cm–1) on Cary Eclipse spectropho-
tometer. The titration experiments were performed by ad-
ding the small amounts of natural 2'-5'A3 or 2'-5'A3-epo-
xy concentrated stock solution, prepared in the same buf-
fer. The final oligoadenylate concentration was 56 �M.
The oligoadenylate-apo-S100A1 solution elliptici-
ty values [�] were corrected by subtracting the corres-
ponding values for natural 2'-5'A3 or 2'-5'A3-epoxy on-
ly and converted into the molar ellipticity [�molar, �], using
the following equation:
[ ] ,
,
�
�
�
�
molar
md
�
100
where �� is the observed ellipticity (degrees) at wave-
length �, m is the molar concentration of protein solu-
tion and d is the path length in centimeters. The secon-
dary structure content of human S100A1 was extracted
using CDNN deconvolution software [19].
NMR Spectroscopy and S100A1 titration with 2'-
5'A3. All NMR data sets were acquired on Varian Unity +
500 NMR spectrometer (1H resonance frequency 500,606
MHz) equipped with three channels, z-gradients unit
and 1H/13C/15N triple resonance probe head with inverse
detection. NMR sample was prepared by dissolving 0.3
mM 15N-labeled human apo-S100A1 protein in 90 %/
10 % H2O/D2O, 20 mM TRIS-d11 and 150 mM NaCl
buffer solution. Spectra were recorded at 293 K using
sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS)
as external indirect reference with coefficients � =
= 0.251449530 and � = 0.101329118 for 13C and 15N
resonance frequencies respectively [20]. In order to ob-
tain the detailed information about the interaction bet-
ween apo-S100A1 and 2'-5'A3, the concentrated stock
solution containing 130 mM of 2'-5'A3 was prepared in
the same buffer. The NMR titration experiment was do-
ne as four consecutive injections (10 �L each) of stock
solution to previously prepared NMR sample. Every ad-
dition was followed by acquiring 2D 1H-15N HSQC spec-
tra. All recorded data were processed with the NMR
Pipe [21] and analyzed with the Sparky [22] software.
Results and discussion. CD spectroscopy. Far-UV
circular dichroism (CD) in the 260–200 nm range is wi-
dely used for the protein structure analysis [23]. In case of
human S100A1, the recorded CD spectrum demonstra-
tes two strong bands at around 222 and 208 nm (Fig. 2,
281
STUDY OF DEPHOSPHORYLATED 2'-5'-LINKED OLIGOADENYLATES IMPACT ON apo-S100A1 PROTEIN
205 210 215 220 225 230 235
-200
-175
-150
-125
2
1
3
4
5
a
Wavelength, nm
�
de
g
cm
2
dm
ol
-1
205 210 215 220 225 230 235
-200
-175
-150
-125
Wavelength, nm
2
�
de
g
cm
2
dm
ol
-1
1
3
4
5
b
Fig. 2. CD spectra of Apo-S100A1 (black/1) in the presence of 8 �M
(red/2), 24 �M (green/3), 40 �M (blue/4) and 56 �M (cyan/5) of
2'-5'A3-nat (a) or 2'-5'A3-epo (b).
a, b), which is an essential feature for the proteins with
high content of �-helical structure [24]. According to
the literature sources, the 3D structure of apo-S100A1
homodimer is dominated by four long �-helices with
two short antiparallel
-sheets in C-terminal parts of
both Ca2+-binding loops [25].
The secondary structure analysis revealed that mo-
re than 50 % of the protein’s secondary structure con-
tent is presented by �-helical elements. The contents of
-turns and randomly coiled elements are very low
(Table). The presented CD spectra are very similar to
those previously obtained in our group or published for
structurally similar S100B protein [26].
An addition of either natural 2'-5'A3 or its epoxy-
modified derivative to the protein solution did not pro-
vide any dramatic changes in initial protein CD spectra
shape. Nevertheless, it is demonstrated that 2'-5'A3 oli-
goadenylates caused tiny alterations in 3D structure re-
flected in a small decrease of molar ellipticity within
both helical bands (Fig. 2, A, B). The analysis of apo-
S100A1 secondary structure demonstrated that addi-
tion of the oligoadenylate at maximum concentration,
either natural or epoxy-modified, led to a decrease in the
�-helical conformation by 6 % (Table). The value of er-
ror was determined by calculating the differences in eva-
luation of the secondary structure content within diffe-
rent regions of the CD spectra by the software.The value
turned out to be around 2 % at the average in all cases.
NMR spectroscopy of human apo-S100A1 protein
and its titration with natural 2'-5'A3 oligoadenylates.
The high-resolution 3D structure of apo-S100A1 is pre-
sented as X-type homodimeric biomolecule. Each su-
bunit has two helix-loop-helix Ca2+-binding EF-hand
motifs of 93 amino acid residues. The spatial structure
S100A1 in solution was solved independently by seve-
ral groups under slightly different conditions (buffer,
pH, ionic strength) and temperatures which could lead
to small differences in Ca2+ affinity [25, 27, 28]. In the
present study, the NMR data sets were acquired on NMR
sample containing 300 �M of 15N-labeled human apo-
S100A1 at pH 7.2 under the same conditions as we ha-
ve used recently [28]. The sequence-specific assign-
ments of 1H and 15N resonances observed on 2D 1H-15N
HSQC spectrum were transferred from previously de-
posited chemical shifts (bmrb code 18089).
A titration curve was obtained after four successive
additions (10 �L each) of 130 mM natural 2'-5'A3 stock
solution prepared in the same buffer. The chemical shift
perturbations (csp) values were calculated from 1H and
15N chemical shifts according to the following equation
[29]:
� � �� � ��
02 2 2. ( ) ( )N H .
An inspection of the 1H and 15N HSQC NMR data
sets exhibited several amino acid residues, which de-
282
SKOROBOGATOV O. Yu. ET AL.
Concentration of
oligoadenylates, � M
2'-5'A3, %a 2'-5'A3-epoxy, %a
�-Helix
-Turn Random coil �-Helix
-Turn Random coil
0 54.5 13.7 21.1 54.5 13.7 21.1
8 53.2 13.9 21.7 52.9 13.9 21.8
24 50.2 14.2 23.1 51.9 14.0 22.4
40 49.8 14.3 23.4 50.6 14.2 23.0
56 48.8 14.4 23.9 49.6 14.3 23.5
Changes in human apo-S100A1 protein’s secondary structure content upon interaction with natural 2'-5' A3 or 2'-5'A3-epoxy
C
h
e
m
ic
a
l
sh
if
t
p
er
tu
rb
ti
o
n
,
p
p
m
Residue number
10 20 30 40 50 60 70 80 90
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Fig. 3. A – chemical shift perturbation (csp) plot on 2D
1
H-
15
N HSQC
spectra upon titration with 2'-5'A
3
(only residues with notable csp
values are presented)
monstrated notable csp values for amide groups (Fig. 3,
A). The recorded experimental data could be used to ob-
tain information about binding constants of native 2'-
5'A3 oligoadenylates to apo-S100A1 [30]. The disso-
ciating constants extracted from the chemical shifts cur-
ves (Fig. 3, B) fall in the moderate 10–5 ... 10–6 M range
suggested that the interaction of 2'-5'A3 with the human
apo-S100A1 protein is biologically relevant, but the
specificity is not very high.
A comparison of the obtained results with known
high-resolution 3D structure of the human apo-S100A1
[27, 28] gave us an idea about structural alterations cau-
sed by 2'-5'A3 binding (Fig. 4). It turned out, that posi-
tions of the residues with higher csp values referred to
the Ca2+-binding loops, which constituted the central
part of the EF-hand domains. Majority of these signals
came from the N-terminal part of the Ca2+-binding mo-
tif (His18, Lys21, As 24, Lys25, Lys30), which was
characterized by strong dependence on tiny changes in
experimental conditions such as temperature, pH or/
and ionic strength of solution. The amino acid residues
within the C-terminal region of the protein globule de-
monstrated lower csp values upon the interaction with
2'-5'A3-nat oligoadenylates: only Val69 and Gln72 re-
vealed notable csp values (Fig. 4). We suggest that even
tiny structural alterations in those regions of the protein
globule may cause changes in its Ca2+-affinity.
The linker region (Glu40-Val51), in addition to C-
terminal helix IV, constitutes another important region of
S100A1 globule, which is mostly responsible for the inter-
action with target proteins/peptides. For instance, amino
acid residues within the linker region are responsible for
the hydrophobic contacts formation in case of interaction
between S100A1 protein and Ryanodine receptor [31].
Another example is the complex between S100A1 and
TRTK12 peptide, derived from actin-capping CapZ pro-
tein, which is formed via the interactions with amino
acids within the «hinge» region (Phe44, Leu45, Lys49)
and helix IV (Ala84, Cys85, Phe88) [32].
We have recently shown that central position in the
linker region is being held by Phe44, which forms the so-
called thiol-aromatic switch that controls hydrophobic
interactions within this part of the protein globule (lin-
ker-helix IV) [28]. Some of the amino acid residues,
located in close proximity to Phe44, demonstrated high
csp values in a response to native 2'-5'A3 binding, name-
ly Thr39, Glu40, and Phe44 itself (Fig. 3, A).
As it has been previously noted, specific structural
changes at the position of Phe44 further propagated to
the whole linker [33]. As a result, notable alterations oc-
curred anywhere within the S100A1 globule. In parti-
cular, the conformational changes might lead to�-helix
shortening by 3–4 residues (one turn) within either the
linker region [28, 33] or helix IV [25, 34]. It has recent-
ly been shown in our group, that similar structural mo-
difications caused the increase of Ca2+ affinity and chan-
ges in cooperativity of Ca2+-binding process [28]. The-
se effects might explain small fluctuations within the
S100A1 �-helical content upon binding natural 2'-5'A3
oligoadenylates and its epoxy derivative (Table).
As has been previously noted, the linker region toge-
ther with helix IV contained several residues strongly con-
served throughout the whole S100 family of proteins
[28]. Comparison of the S100A1 epitops involved in the
interaction process with natural 2'-5'A3 revealed that at
least some of them formed contacts with the target pro-
teins [31, 32]. This supports an idea that oligoadenylates
might regulate interactions of Ca-signaling proteins wi-
thin S100 family with their targets in the similar fashion.
283
STUDY OF DEPHOSPHORYLATED 2'-5'-LINKED OLIGOADENYLATES IMPACT ON apo-S100A1 PROTEIN
Fig. 4. Surface representation of 3D structure of human apo-S100A1
protein (pdb 2LLU) solved at the same experimental conditions [28].
Residues exhibited notable csp are colored as follows: located within N-
terminal (His18, Lys21, Asp24, Lys25) and C-terminal (Val69, Gln72)
Ca
2+
- binding loops are highlighted in yellow and orange, respectively;
residues within linker region and C-terminal part of helix IV (Glu39,
Leu41, Phe44, and Glu91) are colored in red; at the intersubunit in-
terface (Ser2, Glu 3, Ala7, Val54, Tyr74, Thr82, and Asn87) are high-
lighted in green
It worth mentioning, that our experimental data de-
monstrated the impact of 2'-5'A3 on the homodimeric in-
terface within S100A1 globule. Some residues exhibited
higher csp values (Ser2, Glu3, Ala7, Tyr74, Thr82, Asn
87, and Glu91), were involved in the formation of hydro-
phobic intersubunit contacts between helices I/I' and IV/
IV' (Fig. 4). Considering a low value of solvent-acces-
sible surface area of this particular region of the protein
globule, we suppose that 2'-5'A3 does not directly bind to
residues within intersubunit interface, but rather trans-
mits the effect to homodimeric surface upon binding to
Ca2+-binding loop or/and linker region. The similar effect
has previously been reported for structurally similar S100B
protein upon binding Ca2+ ions [35]. The tiny effect of
2'-5'A3 binding to the apo-S100A1 homodimer interfa-
ce might serve as an additional natural element, invol-
ved in the regulation of Ca2+ transduction pathway.
Conclusions. In our study we have demonstrated that
2'-5'A3 can act as a compound, capable of altering the
secondary structure of the human apo-S100A1 protein.
Addition of both natural and epoxy-modified oligoade-
nylates triggered the decrease of its helical structure
nearly to the same extent (around 6 %). We believe that
the changes within its helical structure might trigger
functional Ca2+ signal transduction regulation through
tuning the function of human S100A1 protein. The pre-
sented experimental data obtained by CD and NMR
spectroscopy exhibits tiny conformational alterations
observed within the most important regions of the hu-
man apo-S100A1 globule caused by natural 2'-5'A3 and
its epoxy-modified derivative.
Our results suggested a possible implication of oli-
goadenylates in the regulation of the of S100A1 protein
functioning.
Acknowledgments. Authors would like to acknow-
ledge L. Zhukova for technical assistance with prepara-
tion of different forms of recombinant human apo-
S100A1 protein, Dr. G. Goch for assistance and experti-
se with CD measurements and Dr. I. Dubey for synthe-
sizing the 2'-5'A3-linked oligoadenylates. This work is
partially supported (for IZ) from the Polish National
Centre for Research and Development under research
grant number 178479 (contract number PBS1/A9/13/
2012).
The used equipment was sponsored in part by the
Centre for Preclinical Research and Technology (CePT),
a project co-sponsored by European Regional Deve-
lopment Fund and Innovative Economy, The National
Cohesion Strategy of Poland.
Âèâ÷åííÿ âïëèâó äåôîñôîðèëüîâàíèõ 2'-5' îë³ãîàäåí³ëàò³â
íà êîíôîðìàö³þ á³ëêà apo-S100A1 ìåòîäàìè ãåòåðîÿäåðíîãî
ßÌÐ òà êðóãîâîãî äèõðî¿çìó
Î. Þ. Ñêîðîáîãàòîâ, Ä. Ì. Ëîæêî, ². Þ. Æóêîâ, Î. Â. Êîçëîâ,
Ç. Þ. Òêà÷óê
Ðåçþìå
Íèçüêîìîëåêóëÿðí³ ìåä³àòîðè ïðèðîäíîãî ïîõîäæåííÿ – 2'-5'
îë³ãîàäåí³ëàòè – â³ä³ãðàþòü âàæëèâó ðîëü â àíòèâ³ðóñíîìó ìåõà-
í³çì³, ïîâ’ÿçàíîìó ç ³íòåðôåðîíîì, âîíè ïðè÷åòí³ äî ðîñòó êë³-
òèí, àïîïòîçó òà ³íøèõ âàæëèâèõ ïðîöåñ³â, ùî ïðîò³êàþòü ó êë³-
òèí³. Çâàæàþ÷è íà çíà÷íó êîíöåíòðàö³þ 2'-5' îë³ãîàäåí³ëàò³â âñå-
ðåäèí³ æèâî¿ êë³òèíè, ìîæíà ïðèïóñòèòè, ùî âîíè ñëóãóþòü äî-
äàòêîâèìè á³îëîã³÷íî àêòèâíèìè ñóáñòðàòàìè ³ çäàòí³ ðåãóëþâà-
òè ôóíêö³îíóâàííÿ á³ëêà S100A1 in vivo. Ìåòà. Ïîøóê äîêàç³â ìîæ-
ëèâîñò³ âçàºìî䳿 2'-5' îë³ãîàäåí³ëàò³â ç àïî-ôîðìîþ á³ëêà S100A1
ëþäèíè. Ìåòîäè. Âèêîðèñòàíî ìåòîäè ßÌÐ ³ ÊÄ. Ðåçóëüòàòè.
Îòðèìàí³ äàí³ âêàçóþòü íà òå, ùî âíàñë³äîê âçàºìî䳿 ì³æ 2'-5'
îë³ãîàäåí³ëàòàìè òà S100A1 â³äáóâàþòüñÿ ïåðåáóäîâè ó âòîðèí-
í³é ñòðóêòóð³ îñòàííüîãî. Êð³ì òîãî, âäàëîñÿ âèçíà÷èòè, ÿê³ ñàìå
àì³íîêèñëîòí³ çàëèøêè áåðóòü ó÷àòü ó ö³é âçàºìî䳿. Âèñíîâêè.
Éìîâ³ðíî, 2'-5' îë³ãîàäåí³ëàòè º äîäàòêîâèìè åëåìåíòàìè ñêëàä-
íî¿ ñèñòåìè ðåãóëÿö³¿ ïðîöåñ³â, îïîñåðåäêîâàíèõ ³îíàìè Ñà2+.
Êëþ÷îâ³ ñëîâà: 2'-5' îë³ãîàäåí³ëàòè, S100A1.
Èçó÷åíèå âëèÿíèÿ äåôîñôîðèëèðîâàííûõ 2'-5' îëèãîàäåíèëàòîâ
íà êîíôîðìàöèþ áåëêà apo-S100A1 ìåòîäàìè ãåòåðîÿäåðíîãî
ßÌÐ è êðóãîâîãî äèõðîèçìà
À. Þ. Ñêîðîáîãàòîâ, Ä. Í. Ëîæêî, È. Þ. Æóêîâ, À. Â. Êîçëîâ,
Ç. Þ. Òêà÷óê
Ðåçþìå
Íèçêîìîëåêóëÿðíûå ìåäèàòîðû ïðèðîäíîãî ïðîèñõîæäåíèÿ –
2'-5' îëèãîàäåíèëàòû – èãðàþò âàæíóþ ðîëü â àíòèâèðóñíîì ìå-
õàíèçìå, ñâÿçàííîì ñ èíòåðôåðîíîì, îíè ïðè÷àñòíû ê ðîñòó êëå-
òîê, àïîïòîçó è äðóãèì âàæíûì ïðîöåññàì, ïðîèñõîäÿùèì â
êëåòêå. Ó÷èòûâàÿ êîíöåíòðàöèþ 2'-5' îëèãîàäåíèëàòîâ âíóòðè
æèâîé êëåòêè, ìîæíî ïðåäïîëîæèòü, ÷òî îíè ñëóæàò äîïîëíè-
òåëüíûìè áèî ëîãè÷åñêè àêòèâíûìè ñîåäèíåíèÿìè, ñïîñîáíûìè ðå-
ãóëèðîâàòü ôóíêöèîíèðîâàíèå áåëêà S100A1 in vivo. Öåëü. Ïîèñê äî-
êàçàòåëüñòâ âîçìîæíîñòè âçàèìîäåéñòâèÿ 2'-5' îëèãîàäåíèëà-
òîâ ñ àïî-ôîðìîé áåëêà S100A1 ÷åëîâåêà. Ìåòîäû. Èñïîëüçîâàíû
ìåòîäû ßÌÐ è ÊÄ. Ðåçóëüòàòû. Ïîëó÷åííûå äàííûå óêàçûâàþò
íà òî, ÷òî â èòîãå âçàèìîäåéñòâèÿ ìåæäó 2'-5' îëèãîàäåíèëàòàìè
è áåëêîì S100A1 ïðîèñõîäÿò èçìåíåíèÿ âòîðè÷íîé ñòðóêòóðû ïî-
ñëåäíåãî. Êðîìå òîãî, óäàëîñü îïðåäåëèòü àìèíîêèñëîòíûå îñ-
òàòêè, íåïîñðåäñòâåííî ó÷àñòâóþùèå â ýòîì âçàèìîäåéñòâèè.
Âûâîäû. Âåðîÿòíî, ÷òî 2'-5' îëèãîàäåíèëàòû ÿâëÿþòñÿ äîïîëíè-
òåëüíûìè ýëåìåí òàìè ñëîæíîé ñèñòåìû, ðåãóëèðóþùåé ïðîöåñ-
ñû, îïîñðåäîâàííûå èîíàìè Ñà2+.
Êëþ÷åâûå ñëîâà: 2'-5' îëèãîàäåíèëàòû, S100A1.
284
SKOROBOGATOV O. Yu. ET AL.
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Received 26.02.14
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