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International Journal of Biological Macromolecules 92 (2016) 1288–1297

Contents lists available at ScienceDirect

International  Journal  of  Biological  Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

tructural  and  thermodynamic  studies  of  the  tobacco  calmodulin-like
gs-CaM  protein

odrigo  K.  Makiyamaa,1,  Carlos  A.H.  Fernandesb,1,  Thiago  R.  Dreyerb, Bruno  S.  Modaa,
abio  F.  Matiolib,  Marcos  R.M.  Fontesb,  Ivan  G.  Maiaa,∗

Universidade Estadual Paulista (UNESP), Instituto de Biociências, Departamento de Genética, Botucatu, SP, Brazil
Universidade Estadual Paulista (UNESP), Instituto de Biociências, Departamento de Física e Biofísica, Botucatu, SP, Brazil

 r  t  i  c  l e  i  n  f  o

rticle history:
eceived 25 January 2016
eceived in revised form 8 July 2016
ccepted 7 August 2016
vailable online 8 August 2016

eywords:

a  b  s  t  r  a  c  t

The  tobacco  calmodulin-like  protein  rgs-CaM  is involved  in  host  defense  against  virus  and  is reported  to
possess  an  associated  RNA silencing  suppressor  activity.  Rgs-CaM  is  also  believed  to act  as  an  antiviral
factor  by  interacting  and  targeting  viral  silencing  suppressors  for autophagic  degradation.  Despite  these
functional data,  calcium  interplay  in the  modulation  of rgs-CaM  is still poorly  understood.  Here  we  show
that  rgs-CaM  displays  a  prevalent  alpha-helical  conformation  and  possesses  three  functional  Ca2+-binding
sites. Using  computational  modeling  and  molecular  dynamics  simulation,  we demonstrate  that  Ca2+
a2+-binding protein
almodulin-like protein
gs-CaM

binding  to  rgs-CaM  triggers  expansion  of its  tertiary  structure  with  reorientation  of  alpha-helices  within
the  EF-hands.  This  conformational  change  leads  to the exposure  of a  large  negatively  charged  region  that
may be  implicated  in  the  electrostatic  interactions  between  rgs-CaM  and  viral  suppressors.  Moreover,
the  kd values  obtained  for  Ca2+ binding  to the three  functional  sites  are  not  within  the  affinity  range  of a
typical  Ca2+ sensor.

©  2016  Elsevier  B.V.  All  rights  reserved.
. Introduction

RNA silencing is a potent host defense mechanism against
lant viruses that is counteracted by viral proteins possessing RNA
ilencing suppression activities [1]. Host proteins acting as sup-
ressors of RNA silencing have also been identified and a Nicotiana
abacum calmodulin-like protein called rgs-CaM (for regulator of
ene silencing camodulin-like) was the first whose suppression
ctivity was demonstrated [2]. The tobacco rgs-CaM was  shown
o interact with the helper-component proteinase (HC-Pro), a viral
NA silencing suppressor, and was postulated to be required for HC-
ro-mediated silencing suppression. Consistent with this, Li et al.
3] demonstrated that an rgs-CaM homolog of N. benthamiana is
equired for the RNA silencing suppression activity of �C1, a sup-
ressor encoded by the satellite DNA of Tomato yellow leaf curl China
irus. Contradictory findings, however, have shown that the inter-

ction of rgs-CaM with different viral suppressors has an antiviral
unction [4]. In this case, rgs-CaM is supposed to counteract viral
uppressors by binding to their double-stranded RNA (dsRNA)-

∗ Corresponding author.
E-mail addresses: igmaia@ibb.unesp.br, igmaia1@gmail.com (I.G. Maia).

1 These authors contributed equally to this work

ttp://dx.doi.org/10.1016/j.ijbiomac.2016.08.016
141-8130/© 2016 Elsevier B.V. All rights reserved.
binding domains and targeting them for autophagic degradation.
Moreover, the proposed mechanism requires self-sacrifice of the
rgs-CaM protein. Evolving knowledge indicates that rgs-CaM is an
early-inducible gene implicated in the initial steps of viral infection
[5].

Despite sharing similarity with calmodulins (CaM) and
Calmodulin-like proteins (CML), little is known about the struc-
tural features of rgs-CaM. Such structural studies are important to
shed light on the function of rgs-CaM and also to elucidate its inter-
action with target viral suppressors. As mentioned above, rgs-CaM
was shown to interact with viral suppressors via their positively
charged dsRNA-binding domains [4]. This interaction was reported
to be mediated by electrostatic interactions [4], a distinctive fea-
ture compared to classical CaMs that usually bind to their target
proteins through hydrophobic interactions [6,7]. In this regard, the
crucial roles of electrostatic interactions in the cooperativity of cal-
cium binding and in protein aggregation have been highlighted by
several studies [8–11].

This peculiar characteristic of rgs-CaM in comparison to other
CaMs suggests that rgs-CaM may  exhibit unique structural propri-
eties. In addition, as highlighted by Tadamura et al. [5], rgs-CaM

is the only known CML  protein able to bind to exogenous targets.
In contrast, classical CaMs interact with endogenous targets using
canonical helix-loop-helix EF-hand motifs for Ca2+ binding [12].

dx.doi.org/10.1016/j.ijbiomac.2016.08.016
http://www.sciencedirect.com/science/journal/01418130
http://www.elsevier.com/locate/ijbiomac
http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijbiomac.2016.08.016&domain=pdf
mailto:igmaia@ibb.unesp.br
mailto:igmaia1@gmail.com
dx.doi.org/10.1016/j.ijbiomac.2016.08.016


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R.K. Makiyama et al. / International Journal o

hese EF-hands frequently occur in pairs and most CaMs possess
wo, four or six EF-hands [12].

The structure of CaMs is generally well conserved and presents
- and C-terminal globular lobes linked by a central alpha helix

13]. Each lobe possesses two EF-hand motifs containing a Ca2+-
inding loop composed of a cluster of 12 amino acid residues that
sually starts with an Asp and ends with a Glu. Residues essential
or Ca2+ binding coordination within this cluster are in positions 1,
, 5, 7, 9 and 12, respectively [12,14].

In this study, we examined the Ca2+ binding properties of rgs-
aM in order to learn more about its functionality. For this, the
obacco rgs-CaM was expressed in Escherichia coli and purified.
ubsequently, the binding affinities and possible conformational
hanges associated with Ca2+ binding to recombinant rgs-CaM
ere investigated using different biophysical and computational

pproaches (circular dichroism, isothermal titration calorimetry,
olecular modeling and molecular dynamics simulations).

. Materials and methods

.1. Construction of the rgs-CaM expression vector

The cDNA encompassing the open reading frame of tobacco
gs-CaM (accession number AF329729) was amplified by RT-PCR
sing total RNA extracted from leaves of N. tabacum SR1 and
ene-specific primers supplemented with the restriction sites
or NdeI and XhoI (5′-GGCCATATGTGCATGGAATCAGTTTC-3′ and
′-CTACTCGAGACTTGTCATCATAGCTTTGAAC-3′; sites underlined).
he amplified fragment was gel-purified and cloned into the
deI- and XhoI-digested expression vector pET-28a (Novagen). The

esulting construct was sequenced and subsequently inserted into
. coli BL21(DE3)-CodonPlus-pRIL cells (Stratagene) for expression.

.2. Expression and purification of recombinant his-tagged
gs-CaM

Transformed E. coli cells were grown overnight at 37 ◦C in
uria–Bertani medium supplemented with 100 mg/l kanamicin and
0 mg/l chloramphenicol. The culture was diluted (1:100) in the
ame medium and grown at 37 ◦C to an OD600 of 0.7, followed by
ddition of 1 mM isopropyl-�-d-thiogalactopyranoside (IPTG) and
nduction for 4 h at 28◦C. The harvested cells were resuspended in
ysis buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.25% Tween
0, 1 mM EDTA and 1 mM PMSF) supplemented with lysozyme
1 mg/ml), RNAse A (1 �g/ml) and DNAse I (10 u/ml). After break-
ng the genomic DNA by sonication (6 × 30 s), the suspension was
entrifuged (20.000 × g for 30 min  at 4◦C). The pellets of inclusion
odies were washed twice in buffer A (50 mM Tris–HCl, pH 8.0, and
00 mM NaCl), and solubilized (30 min  at 4◦C) by addition of buffer

 containing 1% sodium dodecyl sulfate (SDS). The recombinant
rotein was then refolded in the presence of 2 M 2-methyl-2,4-
entanediol (MPD) [15,16]. For this, the samples were incubated
t 4◦C for 24 h under gentle agitation. Subsequent purification of
ecombinant His-tagged rgs-CaM (rgs-CaM:His) was performed by
ffinity chromatography using Ni-NTA columns pre-equilibrated
ith buffer A (plus 1% SDS and 2 M MPD) as recommended by the
anufacturer (Qiagen), followed by size exclusion chromatography

sing Superdex 75 10/300 GL columns (GE Healthcare) that were
quilibrated with buffer A containing 2 M MPD. All columns were
inked to a high-performance liquid chromatography system (AKTA
urifier 900; GE Healthcare) and a flow rate of 0.5 ml/min was

sed. Fractions containing recombinant rgs-CaM:His were checked
y standard SDS-PAGE (0.1% SDS, 12% polyacrylamide), stained
ith Coomassie blue, and also by western blot using a monoclonal

nti-polyHistidine antibody (Sigma) and the ECL Western Blot-
gical Macromolecules 92 (2016) 1288–1297 1289

ting Analysis System as recommended by the manufacturer (GE
Healthcare). Protein concentration was  determined by absorbance
at 280 nm in a NanoDrop 2000C spectrophotometer (Thermo
Scientific), using theoretical molecular weights and extinction coef-
ficients provided by the in silico ProtParam tool available on the
ExPASy web  server (http://web.expasy.org/protparam) [17].

2.3. Semi-denaturing mobility-shift assays

The semi-denaturing mobility-shift assay was performed essen-
tially as described [18] using 3 �g of recombinant rgs-CaM in buffer
A (containing 2 M MPD) supplemented with either 5 mM CaCl2 or
5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N’-tetraacetic
acid (EGTA). The samples were incubated at 4◦C for 30 min  and sus-
pended without boiling in sample buffer (62.5 mM Tris–HCl, pH 6.8,
1% SDS, 10% glycerol) devoid of �-mercaptoethanol. Each protein
sample was subsequently electrophoresed on a precast 16% poly-
acrylamide gel containing no SDS (ECL Precast Gel; GE Healthcare).
The gel was stained with Coomassie blue.

2.4. Circular dichroism (CD) spectroscopy

CD measurements were obtained over the spectral range of
200–260 nm using a JASCO J-815 spectropolarimeter (JASCO Spec-
troscopic Co. Ltd., Japan) equipped with a Peltier thermo-controller.
The assays were carried out at 293 K using an optical path length
of 0.5 nm, a scanning speed of 100 nm/min, response time of 1 s,
band width of 2 nm and data pitch of 0.5 nm.  Twenty spectra were
acquired, averaged and corrected for the buffer solution (baseline)
and then normalized to residual molar ellipticity [�]. Previously to
CD measurements, rgs-CaM was  treated with excess of EGTA to
remove possible residual Ca2+ bound to its structure, and the solu-
tion was  subsequently dialyzed against buffer A (containing 2 M
MPD). The effect of Ca2+ in CD spectra was also evaluated by the
addition of 5 mM CaCl2 to the protein sample, in the absence or
presence of 5 mM EGTA. Buffer A plus MPD, CaCl2 and EGTA gave
negligible signals at the concentrations tested and were included
in baseline measurements.

2.5. Isothermal titration calorimetry (ITC)

The thermodynamic parameters of the interaction between rgs-
CaM and calcium ions were determined at 25◦C in buffer A, using
an isothermal titration calorimeter (MicroCal ITC200, GE Health-
care). Prior to the ITC assays, rgs-CaM was  treated with excess EGTA
to remove possible residual Ca2+ bound to its structure, and the
solution was  subsequently dialyzed against the buffer used in the
titration to remove EGTA from the final solution. For a typical Ca2+

titration, 0.2 mM of recombinant rgs-CaM was  placed in the reac-
tion cell (200 �l) and a 10 mM Ca2+ solution was loaded into the
ITC syringe. Each 2.5 min, 2 �l of Ca2+ solution was injected into
the reaction cell. Ca2+ heat of dilution/mixing were determined in
separate control assays and were subtracted from the correspond-
ing titrations. For the stoichiometry (N), dissociation constants (Kd),
enthalpy (�H), and binding-type input parameters were adjusted
to obtain the best fitting model. The values of Kd and �H  were used
to calculate the change in free energy (�G) and entropy (�S) of
Ca2+ binding.

2.6. Modeling and molecular dynamics (MD) simulations

According to the data obtained from the based-threading

method program HHPred (http://toolkit.tuebingen.mpg.de/
hhpred) [19] (score: 169.12; e-value: 2.1e-32; identity: 33%), the
crystal structure at 1.0 Å resolution of a CaM from Paramecium
tetraurelia [Protein Data Bank (PDB) ID: 1EXR] was selected as

http://web.expasy.org/protparam
http://web.expasy.org/protparam
http://web.expasy.org/protparam
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http://web.expasy.org/protparam
http://toolkit.tuebingen.mpg.de/hhpred
http://toolkit.tuebingen.mpg.de/hhpred
http://toolkit.tuebingen.mpg.de/hhpred
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http://toolkit.tuebingen.mpg.de/hhpred


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290 R.K. Makiyama et al. / International Journal o

he best template for initial in silico rgs-CaM structural modeling.
esides, the crystallographic structures of CaMs from Saccha-
omyces cerevisae, Physarum polycephalum,  Entamoeba histolytica,
luyveromyces lactis, Chalamydonas reinhardtii and Homo sapiens
PDB IDs, respectively, 3FWD, 2B10, 4OCI, 4DS7, 3QRX and 2OBH)
ere also identified as putative templates. However, their crystal

tructures presented lower resolution values and shared limited
equence identity with the rgs-CaM primary sequence (lower than
8%).

The initial in silico model of the rgs-CaM structure was gen-
rated using the program MODELLER v.9.10 [20]. Since the first
7 N-terminal residues of rgs-CaM possess no homology with
ny CaM structure deposited in PDB, they were not included in
he model. Calcium ions were modeled at the four putative Ca2+-
inding loops of the rgs-CaM structure and the generated model
called rgs-CaM + Ca-A-B-C-D) was submitted to MD  simulations
sing the program package GROMACS (Groningen Machine for
hemical Simulation) v.4.5.3 [21] in the presence of explicit water
olecules. Protonation states of charged groups were set according

o pH 7.0. Counter ions were added to neutralize the system and
he GROMOS 96 53a6 force field [22] was chosen to perform the

D simulations. The minimum distance between any atom of the
rotein and the box wall was 1.0 nm.

In the next step, four additional models were generated remov-
ng one (rgs-CaM-Ca-A-B-D model), two (rgs-CaM+Ca-A-D), three
rgs-CaM+Ca-C) and four (apo-rgs-CaM) calcium ions from the four
utative Ca2+-binding loops as found in the CaM of P. tetraurelia
sed as model. The letters A, B, C and D denote the presence of
alcium in the corresponding Ca2+-binding loops. For all models,
nergy minimization (EM) using steepest descent algorithm was
erformed to generate the starting configuration of the system.
fter this step, 200 ps of MD  simulation with position restraints
pplied to the protein (PRMD) was executed to gently relax the sys-
em. All the MD  simulations were carried out in a periodic truncated
odecahedron box under constant temperature (298 K) and pres-
ure (1.0 bar) maintained by the coupling to an external heat and an
sotropic. Then, 200 ns for the rgs-CaM+Ca-A-B-D model and 100 ns
or the remaining models of unrestrained MD simulations were cal-
ulated to evaluate the stability of all the structures generated. The
verall quality of all in silico models obtained after MD simulation
as checked using RAMPAGE [23] and ProSA-web (https://prosa.

ervices.came.sbg.ac.at/prosa.php). The average root mean square
uctuations (RMSF) of the backbone atoms from the final models
ere calculated and converted to B-factor values using GROMACS

.4.5.3 [21] (Suppl. Fig. 1). The same program was used to calculate
he radius of gyration (Rg) of each model during MD  simulation.
he electrostatic potential surfaces of the in silico models were gen-
rated by APBS (Adaptive Poisson-Boltzmann Solver) electrostatic
alculations at 150 mM NaCl with a probe size of 1.4 Å on Chimera
.1.9 [24], after the conversion of the PDB file into a PQR file (pH
.0) using the online server PDB2PQR with the PARSE force field
25]. All structural figures were generated using Chimera v.19 [24]
nd PyMOL v.1.3 [26].

. Results

.1. Expression and purification of soluble rgs-CaM

Expression of the His-tagged rgs-CaM in E. coli resulted in a
olypeptide with an apparent molecular mass of 21 kDa (Fig. 1A),
hich was present in the insoluble inclusion body fraction (Fig. 1B).

fter several unsuccessful attempts of solubilization in nonde-
aturing conditions, the inclusion bodies were solubilized using
DS and the recombinant protein was refolded in the presence
f the amphipathic solvent MPD. MPD  has been successfully used
gical Macromolecules 92 (2016) 1288–1297

in the refolding of SDS-denatured proteins avoiding disruption of
the protein structure [15,16]. After refolding, the recombinant rgs-
CaM was  further purified using a combination of metal affinity
chromatography on a Ni-NTA column and size exclusion chro-
matography (Fig. 1C). Purification resulted in a single band of
22 kDa, visible on SDS-PAGE, which is close to the estimated molec-
ular mass of native rgs-CaM (21 kDa). Purified rgs-CaM was further
recognized by a His-tag monoclonal antibody in western blot anal-
ysis, thus confirming correct fusion. It should be noted that the
His-tag was  not removed from purified rgs-CaM for subsequent
analyses.

3.2. Secondary structural predictions using CD spectroscopy

The structural integrity of the refolded rgs-CaM was checked
using CD spectroscopy. The resulting CD spectrum displayed min-
imum values around 208 and 222 nm,  indicating a structured
conformation (Fig. 2A). These two minima are typically observed in
proteins with significant alpha-helical content, which is the case of
plant CaMs [27]. To characterize a possible conformational change
of rgs-CaM upon Ca2+ binding, CD spectra were further recorded in
the presence of 5 mM CaCl2 and after the addition of 5 mM EGTA.
As shown in Fig. 2B, the addition of Ca2+ to rgs-CaM promoted a
slight increase in molar ellipticity at 222 nm.  In contrast, no spec-
tral changes were detected in the presence of EGTA as compared to
purified rgs-CaM (apo-rgs-CaM). A modest effect on the CD spec-
trum after Ca2+ addition was  also observed for two  CMLs from
Arabidopsis thaliana (designated CML42 and CML43) [28,29]. How-
ever, for both proteins, the observed effect was  attributed to a helix
reorientation rather than to an increase in alpha helix content after
Ca2+ binding [28,29].

3.3. Calcium promotes a shift in rgs-CaM mobility

Calcium ions have been reported to alter the electrophoretic
mobility of Ca2+-binding proteins by promoting faster migration
compared to proteins maintained in a Ca2+-free state [30,31]. We
therefore tested the effect of Ca2+ on the migration of recombinant
rgs-CaM using a semi-denaturing mobility-shift assay. When CaCl2
was added to rgs-CaM just before electrophoresis, a faster migra-
tion of CaCl2-treated rgs-CaM as compared to purified rgs-CaM was
noted (Fig. 3). In contrast, no shift was observed when EGTA was
added to the protein sample containing 5 mM CaCl2. In this case,
the EGTA-treated rgs-CaM migrated similarly to purified rgs-CaM.

3.4. ITC results indicate the presence of three Ca2+-binding sites
in rgs-CaM

A typical set of ITC data for Ca2+ interaction with rgs-CaM is
shown in Fig. 4. Higher and lower panels show the raw calorimetric
data for the Ca2+-into-rgs-CaM titration and the binding isotherm,
respectively. Based on primary sequence analyses (Fig. 5), a sequen-
tial four-binding site model was used to fit ITC data as previously
described [29,32,33]. In this regard, the resulting calorimetry data
was best fitted by the three Ca2+-binding site model (Table 1 and
Fig. 4), which presented the lower chi-square value (128.8) com-
pared to the two- and four-binding site models (162.0 and 198.8,
respectively). This is in line with a previous assumption suggest-

ing the existence of only three Ca2+-binding sites in rgs-CaM [2].
It should also be emphasized that the interaction of rgs-CaM with
Ca2+ displayed a negative enthalpy change, indicative of an exother-
mic  binding event (Table 1).

http://https://prosa.services.came.sbg.ac.at/prosa.php
http://https://prosa.services.came.sbg.ac.at/prosa.php
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http://https://prosa.services.came.sbg.ac.at/prosa.php
http://https://prosa.services.came.sbg.ac.at/prosa.php


R.K. Makiyama et al. / International Journal of Biological Macromolecules 92 (2016) 1288–1297 1291

Fig. 1. Expression in E. coli and purification of recombinant tobacco rgs-CaM. A) Total cell extracts of non-induced (lanes NI) or IPTG-induced (lanes I) bacteria expressing
r rgs-Ca
c on ch
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gs-CaM (different clones are shown). B) Analysis of the solubility of recombinant 

entrifugation of sonicated cells. C) Purified His-tagged rgs-CaM after size exclusi
tandard in kDa (PageRuler Plus Ladder 10–250 kDa; Fermentas). Gels were stained

.5. Primary sequence comparisons and homology modeling
ighlights the presence of three Ca2+-binding sites in rgs-CaM

A search for proteins structurally related to rgs-CaM using the
Hpred server revealed structural similarity to CaMs from K. lactis

PDB ID: 4DS7), P. tetraurelia (PDB ID: 1EXR) and H. sapiens (PDB
D: 3OX6). Inspection of the primary structure alignment (Fig. 5A)

evealed that only three out of the four putative Ca2+-binding loops
f rgs-CaM (indicated as A, B and D) have a canonical 12-residue
a2+-binding loop sequence signature [D-X-Y-X-Y-G-X-�-X-X-X-
,  where Y indicates either D or N residues, and � indicates a
M. Ins and Sol denote insoluble and soluble fractions, respectively, resulting from
romatography. All samples were subjected to 12% SDS-PAGE. M-  Molecular mass
Coomassie Brilliant Blue.

hydrophobic residue] [12]. On the other hand, loop C showed no
conservation of D/N residues at positions 1, 3 and 5, and of E at posi-
tion 12, therefore suggesting possible non-functionality (Fig. 5A).
Further comparative information about these loops is provided by
the alignment (Fig. 5B) of rgs-CaM with CML42 and CML43 from A.
thaliana that also possess only three Ca2+-binding sites [28,29].

To obtain further structural insights into the Ca2+ coordina-

tion of rgs-CaM, we  generated in silico models for apo-rgs-CaM
and for rgs-CaM loaded with four (rgs-CaM+Ca-A-B-C-D), three
(rgs-CaM+Ca-A-B-D model), two  (rgs-CaM+Ca-A-D model) and one
(rgs-CaM+Ca-C model) Ca2+ ions (Table 2; Fig. 6A and B). The in sil-



1292 R.K. Makiyama et al. / International Journal of Biological Macromolecules 92 (2016) 1288–1297

Fig. 2. Far-UV circular dichroism spectra of recombinant rgs-CaM in apo state
(black), in the presence of 5 mM CaCl2 (red) and after subsequent addition of 5 mM
EGTA (blue). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

Fig. 3. Effects of Ca2+ on the electrophoretic mobility of recombinant rgs-CaM.
The protein samples analyzed by CD (as described in the legend of Fig. 2) were
subjected to PAGE (16%) under semi-denaturing conditions. A faster migration of
rgs-CaM in the presence of CaCl2 (5 mM)  was observed. M:  Molecular mass stan-
dard in kDa (PageRuler Plus Ladder 10–250 kDa; Fermentas). The gel was  stained
with Coomassie Brilliant Blue.

Fig. 4. Isothermal calorimetric data of Ca2+ binding to rgs-CaM. The top panel shows
the raw power output (�cal/s) per unit time (min). Bottom panel shows the inte-
grated data (kcal/mol of injectant versus molar ratio of Ca2+ to rgs-CaM). These data
were obtained from the raw power output as the area underneath each peak, which
is  then corrected for baseline heat injections and Ca2+ dilution heat and mixing. The
solid line represents the best fit of the data to the sequential 3 binding site model.

Table 1
Thermodynamic parameters of Ca2+-binding to rgs-CaM obtained from ITC analysis.

Binding Site Kd (M)  �H (kcal/mol) �S (cal/mol/deg)

1 (1.27 ± 0.07) x 10−4 −2.10 ± 0.07 10.8
−5
2  (7.87 ± 0.42) x 10 0.32 ± 0.09 19.8

3  (1.02 ± 0.05) x 10−2 −31.61 ± 1.31 −96.9

ico models confirmed that rgs-CaM, as classical CaMs, has N- and
C-terminal globular lobes (with two  putative Ca2+-binding loops
each) separated by a central helix of 26 residues (Fig. 6). More-
over, all models showed an overall good quality, as evaluated by
the distribution of residues in favored and allowed regions of the
Ramachandran plot and by Z-score of ProSA-web [34] (Table 2).
These models were deposited in the ModelArchive public database
[35], and their Digital Object Identifier (DOI) are available in Table 2.

Interestingly, along the MD simulations of the rgs-CaM+Ca-A-B-
C-D model, all Ca2+ ions remained coordinated in their loops, with
the exception of the one present in loop C (Fig. 6A). The same feature
was observed when Ca2+ was  loaded only on loop C (rgs-CaM+Ca-C
model), suggesting that this loop is unable to coordinate Ca2+. These
results are consistent with the aforementioned sequence alignment

predictions and ITC data, thus indicating that rgs-CaM, unlike clas-
sical CaMs, has only three sites of Ca2+ coordination, namely loops
A, B and D.



R.K. Makiyama et al. / International Journal of Biological Macromolecules 92 (2016) 1288–1297 1293

Table  2
Overall quality, radius of gyration (Rg) and Digital Object Identifiers of rgs-CaM in silico models deposited on ModelArchive public database.

Model Z-scorea Residues in allowed and favored regions (%)b Rg (nm)c DOId

apo-rgs-CaM −7.4 100 1.7 ma-asrjx
rgs-CaM+Ca-A-B-C-D −5.6 98.6 2.3 ma-a9cbt
rgs-CaM+Ca-A-B-D −5.4 96.4 2.3 ma-awfwa
rgs-CaM+Ca-A-D −6.1 98.9 2.2 ma-a2nix
rgs-CaM+Ca-C −4.7 99.3 1.8 ma-aborg

a Calculated by ProSa-web [27].
b According to Ramachandra plot calculated by RAMPAGE [17].
c Calculated by GROMACS v.4.5.3 [15].
d Digital object identifier (DOI) of the in silico structure deposited on ModelArchive [33] public database.

Fig. 5. Multiple amino acid sequence alignments. (A) Alignment of rgs-CaM with homologous structures identified by the HHpred server. K lac CaM: Calmodulin from K.
lactis  NRRL Y-1140 (PDB ID 4DS7); P tet CaM: Calmodulin from P. tetraurelia (PDB ID 1EXR) and H sap CaM: Human calmodulin (PDB ID 3OX6); (B) Alignment of rgs-CaM
with  CML42 (GI: 75337714) and CML43 (GI: 75333888) from A. thaliana. The putative Ca2+ binding-loops are designated A, B, C and D. Red boxes highlight the amino acids of
the  canonical 12-residue Ca2+-binding loops seen in CaM [D-X-Y-X-Y-G-X-�-X-X-X-E, where Y indicates D/N residues, and � indicates a hydrophobic residue]. The green box
h . Supe
i  the re
t

3
e
n

a
[
i

ighlights the flexible region of the central helix observed in Ca2+-bound structures
n  loops A and D of rgs-CaM in relation to the CML  sequences. (For interpretation of
his  article.)

.6. In silico models of Ca2+-bound rgs-CaM detected an
xpansion on its tertiary structure with exposition of a large
egatively charged surface

It has been previously reported that Ca2+-bound CaMs have

 more expanded tertiary structure than their apo counterparts
13,36]. In this regard, we found that the removal of all Ca2+ resulted
n a more compact rgs-CaM model compared to the one loaded
rior arrows in panel B indicate the hydrophobic amino acid substitutions observed
ferences to colour in this figure legend, the reader is referred to the web  version of

with three Ca2+. This observation was  well correlated with the
Rg values of both apo and four calcium-containing rgs-CaM mod-
els (1.7 nm versus 2.3 nm;  Table 2). Thus, the loading of Ca2+ into
loops A, B and D promoted an expansion of the tertiary structure
of rgs-CaM, as already reported for CaMs [37]. C� superposition of

the central helix of the rgs-CaM+Ca-A-B-C model over the central
helix of the apo-rgs-CaM model clearly illustrates this expan-
sion. In this case, a different reorientation of the EF-hand helices



1294 R.K. Makiyama et al. / International Journal of Biological Macromolecules 92 (2016) 1288–1297

Fig. 6. In silico modeling of rgs-CaM. (A) Cartoon representation of the final rgs-CaM model with Ca2+ modeled in the four predicted Ca2+-binding loops (rgs-CaM+Ca-A-B-C-D
model). The residues predicted to coordinate Ca2+ in rgs-CaM are represented by sticks. Along the MD simulations, the Ca2+ in loop C escaped from the loop whereas the
other  ions remained coordinated in the other loops. The flexible region near the middle of the central helix (E120-E125) is also highlighted. (B) Cartoon representation
of  C� superposition of the central helix of rgs-CaM+Ca-A-B-D (green) over apo-rgs-CaM (yellow) showing the expansion of rgs-CaM tertiary structure and the different
reorientations of the helices within the EF-hands upon Ca2+ binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version  of this article.)

Fig. 7. Electrostatic potential surfaces of the in silico models. (A) apo-rgs-CaM and (B) rgs-CaM+Ca-A-B-D. Both structures are shown in the same orientation after C�
superposition. The electrostatic potential surfaces were generated by APBS (Adaptive Poisson-Boltzmann Solver). Red and blue colored regions denote negative and positive
charges,  respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



R.K. Makiyama et al. / International Journal of Biological Macromolecules 92 (2016) 1288–1297 1295

Fig. 8. Structural comparisons between the P. tetraurelia CaM (PDB ID 1EXR) and the rgs-CaM + Ca-A-B-D model. (A) Cartoon representation of crystallographic structure of P.
tetraurelia CaM with hydrophobic residues (Ala, Ile, Leu, Phe, Pro and Val) exposed to the solvent after Ca2+-binding (highlighted as yellow sticks). (B) Cartoon representation
of  the rgs-CaM+Ca-A-B-D model with hydrophobic residues exposed to solvent after Ca2+-binding (highlighted as yellow sticks). The hydrophobic residues buried into the
N ntatio
a ). The
t le.)

b
(
b
t
(

l
C
(
i
t
t
c
s
i
d
a
o
a
o

n
t
h
I

-terminal globular lobe are highlighted as green olive sticks. (C) Cartoon represe
nd  Thr) exposed to the solvent after Ca2+-binding (highlighted as light green sticks
o  colour in this figure legend, the reader is referred to the web version of this artic

etween apo- and calcium-containing models could be observed
Fig. 6B). Interestingly, when Ca2+ was loaded into loops A and D
ut not B (rgs-CaM-A-D model), rgs-CaM adopted an expanded ter-
iary structure conformation showing an Rg value around 2.3 nm
Table 2).

Along the MD  simulations of the rgs-CaM models with Ca2+

oaded into, at least, loops A and D (rgs-CaM+Ca-A-B-D and rgs-
aM+Ca-A-D models), a region near the middle of the central helix
E120 through E125 as denoted in Fig. 5A) presented great mobil-
ty, adopting even a disordered secondary structure at the end of
he majority of the simulations (Fig. 6A). This flexibility confers
o the central helix a bendable joint on its center that probably
ontributes to the reorientation of the EF-hands to become more
olvent-exposed in the Ca2+-bound models (Fig. 6B). In contrast,
n MD  simulations of apo-rgs-CaM, this region remained stable
uring all 100 ns simulations. The deduced amino acid sequence
lignments revealed that the E120-E125 region of rgs-CaM is anal-
gous to the K77-S81 region of classical CaMs (Fig. 5A), which is
lso located in the middle of the central helix and has a high degree
f mobility as previously shown by NMR  assays [37].

Another typical feature of classical CaM is the exposure of a sig-

ificant amount of non-polar surface area upon expansion of its
ertiary structure [37,38]. This solvent-exposed area results in a
ydrophobic pocket that serves as a target interaction site [32].

n this context, Nakahara et al. [4] reported previously that rgs-
n of the rgs-CaM+Ca-A-B-D model with negatively charged residues (Asp, Glu, Ser
 figure was generated using PyMOL v.1.3 [22]. (For interpretation of the references

CaM can interact with the 2b suppressor of Tomato aspermy virus
by electrostatic interactions, thus contrasting with classical CaMs
that usually use hydrophobic patches to interact with their targets
[6,7]. In fact, a comparative analysis of the electrostatic potential
surfaces of the apo-rgs-CaM and rgs-CaM+Ca-A-B-D models in the
same orientation revealed that the Ca2+-induced conformational
expansion leads to the exposure of a negatively charged region
(Fig. 7). This anionic area is located mainly on the N-terminal globu-
lar lobe and is complemented by some portions of the central helix
and C-terminal lobe, thus forming a large and negatively charged
pocket. A careful comparison between the crystallographic struc-
ture of the Ca2+-bound CaM from P. tetraurelia (PDB ID: 1EXR) and
the rgs-CaM+Ca-A-B-D model reveals a different distribution of
the conserved hydrophobic residues on these structures (Fig. 8).
In the CaM from P. tetraurelia,  the surface-exposed hydrophobic
residues are located mainly on the N-terminal globular lobe and
in some portions of the central helix and C-terminal lobe (Fig. 8A).
In contrast, the corresponding regions of the rgs-CaM+Ca-A-B-D
model have fewer solvent-exposed hydrophobic residues and con-
tain several negatively charged residues (Fig. 8C). This is especially
true for the N-terminal lobe in which the majority of the residues

are buried into the EF-hand structure (Fig. 8B). These negative
residues comprise the anionic pocket that could be responsible for
the interaction of rgs-CaM with the dsRNA-binding domains of viral
suppressors.



1 f Biolo

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296 R.K. Makiyama et al. / International Journal o

. Discussion

Rgs-CaM is a CaM-related protein involved in the dynamic inter-
lay between viruses and plants. Conflicting results from different
roups suggest that rgs-CaM could target viral suppressors for
utophagic degradation [4] or interact with them having a posi-
ive effect on suppressor activity [3]. Earlier homology modeling
tudies demonstrated that rgs-CaM possesses two predicted EF-
and motifs separated by a negatively charged cleft [4]. Despite
he presence of these predicted EF-hands, the ability of rgs-CaM to
ind Ca2+ and its effective role as a Ca2+ sensor remain elusive.

In this regard, our CD spectral data revealed that rgs-CaM is
ensitive to Ca2+ binding, but only modest effects on its overall sec-
ndary structure are detected upon binding (Fig. 2). Our results
sing EGTA in CD measurements and mobility shift assays showed
hat Ca2+ binding to rgs-CaM is a reversible process. Moreover, by
enerating structural in silico models for calcium-free and calcium-
ound rgs-CaM (Fig. 6B; Table 2), we found that rgs-CaM structure
ndergoes a calcium-induced expansion and reorientation of the
lpha-helices within the EF-hands (Fig. 6B; Table 2). This confor-
ational change is commonly observed in classical CaMs [37]. The

eorientation of the helices leads to exposure to the solvent of a
arge EF-hand area that could be responsible for the slight increase
n molar ellipticity at 222 nm detected upon Ca2+ addition to rgs-
aM (Fig. 2), as previously observed for CML43 [28]. Additionally,

n our Ca2+-bound rgs-CaM models, a higher flexibility near the
iddle of the central helix along the MD  simulations was noted, a

eature that probably contributes to the EF-hand rearrangements.
Our ITC data indicate that rgs-CaM has only three functional

a2+-binding sites. This was further supported by the information
leaned from amino acid sequence predictions and by inspection
f the rgs-CaM models generated in silico. Collectively, our results
ndicate that loop C in the C-terminal EF-hand of rgs-CaM is unable
o bind calcium. Remarkably, this loop shows no conservation of the
esidues typically found in canonical Ca2+-binding loops of classical
aMs (Fig. 5A). Bender et al. [28] reported that A. thaliana CML43,
hich acts as a Ca2+ sensor, also possesses only three functional
a2+-binding loops. One of them has high Ca2+ affinity [dissocia-
ion constants (kd) of 8.16 nM]  and is supposed to play a structural
ole, whereas the two remaining loops have moderate Ca2+ affin-
ty (kd = 4.85 �M)  and are supposed to be regulatory. In contrast to
gs-CaM, the non-functional loop of CML43 (loop B) is located in
he N-terminal EF-hand and shows no conservation of the residues
nvolved in Ca2+ coordination (Fig. 5B).

According to the ITC results, rgs-CaM has two Ca2+-binding
ites (sites A and D) with moderate Ca2+ affinity (kd = 78.7 �M and
27 �M,  sites 1 and 2 in Table 1), and a third one (B) with low
a2+ affinity (kd = 10.2 mM;  site 3 in Table 1). This moderate/low
a2+ affinity may  be attributed to the substitution of conserved
ydrophobic residues within the EF-hands, especially in loops A
nd D (Fig. 5B). Surprisingly, despite the fact that rgs-CaM possesses
everal features of a Ca2+ sensor, as indicated by our structural data,
he kd values determined by ITC are not within the affinity range of

 typical Ca2+ sensor (1–0.1 �M)  [12]. This fact, however, does not
reclude the role of rgs-CaM as a sensor since its Ca2+ affinity may
e increased in the presence of its target viral suppressor. This phe-
omenon has already been reported for other known CaMs [12]. In
ammals, for example, the affinity of CaM for Ca2+ was increased

5-fold in the presence of its interacting protein [38]. Interestingly,
asal cytosolic levels of Ca2+ (about 100 nM)  [12,39] were insuffi-
ient to induce important structural changes in rgs-CaM. It seems
herefore likely that the observed expansion in rgs-CaM structure

ould be triggered by transitory increases in cytosolic Ca2+ lev-
ls as would be expected during pathogen attack. Consistent with
his, previous studies have shown that rgs-CaM is widely expressed
mmediately after wounding [5], a stress that occurs simultane-
gical Macromolecules 92 (2016) 1288–1297

ously to virus invasion and is also reported to alter Ca2+ homeostasis
[40].

Of note, the thermodynamic results relative to sites A and D
(sites 1 and 2 in Table 1), revealing small enthalpy and large positive
entropy changes, corroborate the observed Ca2+-dependent con-
formational modification [12]. Concerning site B (site 3 in Table 1),
despite its low Ca2+ affinity, this site showed a large and exother-
mic  enthalpy and an unfavorable binding entropy in calorimetric
studies. This is consistent with the large and exothermic enthalpy
observed for Ca2+-binding of calbindin D9k due to the dehydration
of the EF-hands [11]. Most noteworthy is the fact that the absence
of Ca2+ in site B has no impact on the expansion of the rgs-CaM ter-
tiary structure. Accordingly, rgs-CaM adopted the same expanded
conformation when Ca2+ was loaded into sites A and D or into the
three predicted Ca2+-binding sites.

Despite corroborating previous findings suggesting that rgs-
CaM interacts with its targets by electrostatic interactions [4], our
results provide additional information about peculiar aspects of
this protein. Most importantly, we observed that the binding of
two Ca2+ to rgs-CaM is sufficient to promote surface exposure of a
large negatively charged pocket, which might be responsible for its
interaction with the dsRNA-binding domains of viral suppressors.

5. Conclusions

The results obtained indicate that rgs-CaM has three puta-
tive Ca2+-binding sites. However, Ca2+ binding to the two sites
possessing higher Ca2+ affinity is sufficient to trigger a conforma-
tional change in an expanded structure with reorientation of the
helices within the EF-hands. In addition, our structural data support
the idea that rgs-CaM interacts with its targets through electro-
static interactions, thus contrasting classical CaMs that usually use
hydrophobic patches [6,7]. It seems plausible that these negatively
charged residues comprised within an anionic pocket might be
responsible for the interaction of rgs-CaM with the dsRNA-binding
domains of viral suppressors.

Author contributions

Conceived and designed the work: RKM, CAHF, MRMF  and IGM.
Performed the experiments: RKM, CAHF, TRD, BSM and FFM. Wrote
the manuscript: MRMF  and IGM with feedback from CAHF.

Acknowledgments

R. K. Makiyama was  recipient of a PhD fellowship from FAPESP
(2010/03001-0). C. A. H. Fernandes is recipient of a post-doctoral
fellowship from FAPESP (2013/17864-8). I. G. Maia and M.  R. M.
Fontes are recipients of research fellowships from CNPq. Com-
putational resources were supplied by the Center for Scientific
Computing (NCC/GridUNESP) of the São Paulo State University
(UNESP).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2016.
08.016.

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