Starch-binding domains in the CBM45 family – low-affinity
domains from glucan, water dikinase and a-amylase
involved in plastidial starch metabolism
Mikkel A. Glaring
1,2
, Martin J. Baumann
1
, Maher Abou Hachem
1
, Hiroyuki Nakai
1
, Natsuko Nakai
1
,
Diana Santelia
3
, Bent W. Sigurskjold
4
, Samuel C. Zeeman
3
, Andreas Blennow
2
and Birte Svensson
1
1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark
2 VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of
Copenhagen, Frederiksberg, Denmark
3 Department of Biology, ETH Zu
¨
rich, Switzerland
4 Department of Biology, University of Copenhagen, Denmark

sentatives of each of the two classes of enzyme carrying CBM45-type
domains, the Solanum tuberosum a-glucan, water dikinase and the Arabid-
opsis thaliana plastidial a-amylase 3, were expressed as recombinant pro-
teins and characterized. Differential scanning calorimetry was used to
verify the conformational integrity of an isolated CBM45 domain, reveal-
ing a surprisingly high thermal stability (T
m
of 84.8 °C). The functionality
of CBM45 was demonstrated in planta by yellow ⁄ green fluorescent protein
fusions and transient expression in tobacco leaves. Affinities for starch and
soluble cyclodextrin starch mimics were measured by adsorption assays,
surface plasmon resonance and isothermal titration calorimetry analyses.
The data indicate that CBM45 binds with an affinity of about two orders
of magnitude lower than the classical starch-binding domains from extra-
cellular microbial amylolytic enzymes. This suggests that low-affinity
starch-binding domains are a recurring feature in plastidial starch metabo-
lism, and supports the hypothesis that reversible binding, effectuated
through low-affinity interaction with starch granules, facilitates dynamic
regulation of enzyme activities and, hence, of starch metabolism.
Abbreviations
AMY3, a-amylase 3; AtAMY3, Arabidopsis thaliana a-amylase 3; CBM, carbohydrate-binding module; DSC, differential scanning calorimetry;
GWD, a-glucan, water dikinase; IPTG, isopropyl thio-b-
D-galactoside; ITC, isothermal titration calorimetry; PWD, phosphoglucan, water
dikinase; SBD, starch-binding domain; SPR, surface plasmon resonance; StGWD, Solanum tuberosum a-glucan, water dikinase;
TEV, tobacco etch virus; YFP ⁄ GFP, yellow ⁄ green fluorescent protein.
FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1175
branch points. Together, these polymers are laid down
as alternating semicrystalline and amorphous layers to
form the supramolecular granule structure. The semi-
crystalline layers are made up primarily of packed

EC 3.2.1.1). Two types of CBM45-containing GWD
have been identified in plants, one of which is plastid-
ial and essential for normal starch metabolism
(GWD1 ⁄ R1 ⁄ SEX1) [8,9]. The second GWD, called
GWD2 in Arabidopsis, is extraplastidial and has no
apparent role in starch degradation [10]. The plastidial
a-amylase AMY3 is not required for normal transitory
starch metabolism in Arabidopsis [11], but a functional
role in planta has been inferred from knock-out studies
in phosphoglucan phosphatase (SEX4) and quadruple
debranching enzyme (ISA1 ⁄ ISA2 ⁄ ISA3 ⁄ LDA) mutant
backgrounds [12,13]. In both enzyme classes, the
CBM45s are present as N-terminal tandem domains,
separated by a linker domain of varying length. No
three-dimensional structure is available for CBM45,
but a recent bioinformatic analysis produced a rough
model and identified two tryptophan residues as puta-
tive binding sites in the N-terminal CBM45 from Ara-
bidopsis GWD1 [14]. These two tryptophans have
previously been experimentally confirmed as pivotal
for the starch-binding ability of potato GWD [7].
The metabolism of plastidial starch in leaves of
plants is a tightly regulated process. The available pho-
tosynthate has to be balanced with the energy and car-
bohydrate needs of the plant during the subsequent
dark period. Perturbations in this process lead to
severe phenotypes and retardation of growth [15,16].
Plastidial starch metabolism has been well character-
ized in the model plant Arabidopsis thaliana, and
numerous enzymes are involved in the process of

Identification and bioinformatic analysis of
CBM45s from GWD and AMY3
Family CBM45 sequences were obtained from the car-
bohydrate-active enzymes database (CAZY, http://
www.cazy.org). Furthermore, a search of the translated
nucleotide database at the National Center for Bio-
technology Information ()
uncovered several additional sequences with homology
to StGWD and AtAMY3, which were included in the
Starch-binding domains in the CBM45 family M. A. Glaring et al.
1176 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS
analysis. Alignment of these sequences showed not
only extensive conservation in the catalytic domains,
suggesting a preservation of function, but also the exis-
tence of the N-terminally appended CBM45 domains
(data not shown). The sequences used in the subse-
quent analysis included CBM45s from 45 proteins
from primarily photosynthetic eukaryotes (plants,
green algae and red algae), as well as from apicom-
plexan parasites (Doc. S1). A common characteristic
of these organisms is the presence of starch or starch-
like crystalline polysaccharides. The starch synthesis
ability of the apicomplexan parasites is believed to be
derived from an endosymbiosis of a red alga, with
following loss of photosynthetic capacity [29].
The CBM45s are present as tandem domains in the
N-terminal part of StGWD and AtAMY3 and sepa-
rated by a linker of approximately 200 and 50 amino
acids, respectively (Fig. 1A). The alignment of all iden-
tified CBM45s revealed that each contains five aro-

formed a separate, mixed group, reflecting the evolu-
tionary distance and low homology between these
sequences. Overall, it appears as though CBM45s and
the tandem structure of these domains arose early in
evolution, perhaps in an ancestor of the current photo-
synthetic eukaryotes. It has been proposed that GWD
sequences were a prerequisite for the appearance of
semicrystalline starch-like polymers [29]. If this is
indeed the case, the appended CBM45 domains could
represent a truly ancient SBD and, perhaps, be one of
the first CBMs dedicated to starch binding.
Expression and purification of CBM45s from
potato GWD and Arabidopsis AMY3
In order to characterize the CBM45s from StGWD,
several expression constructs were produced and
tested. Because there is no structural information on
CBM45, putative domain borders were assigned on the
basis of the predicted secondary structure and homol-
ogy to other CBMs. Thirteen constructs with an N-ter-
minal tobacco etch virus (TEV) protease-cleavable
Histidine (His)-tag, containing both the single and
A
B
Fig. 1. Overview of the CBM45 domains.
(A) Domain structure of StGWD and
AtAMY3 showing the chloroplast transit
peptide (TP, black), tandem CBM45s (grey)
and catalytic domain (light grey). The size
of the proteins is given in amino acids (aa).
(B) Sequence alignment of CBM45 domains

native full-length GWD.
Initial differential scanning calorimetry (DSC)
screening of the His-tagged StGWD CBM45-2A indi-
cated a high unfolding temperature. This protein gave
rise to a broad asymmetric thermogram with
T
m
= 78.1 °C (data not shown). Proteolytic removal
of the N-terminal His-tag yielded a symmetric single
peak thermogram with T
m
= 84.8 °C at pH 8.0. Inter-
estingly, the unfolding was partially reversible, as dem-
onstrated by the 83% and 74% area recovery of the
second and third scans, respectively (Fig. 2). Fitting a
two-state model to the reference- and baseline-corrected
calorimetric trace resulted in an excellent fit, yielding
DH = 414.1 ± 0.7 kJÆmol
)1
, attesting to the high ther-
mal stability and conformational integrity of CBM45-
2A from StGWD. This CBM displayed similarly high
conformational stability at pH 7.0 (T
m
= 87.1 °C), but
the reversibility was decreased significantly and the pro-
tein was prone to aggregation (data not shown). The
reason for this extraordinarily high stability is
unknown, but it strongly suggests that CBM45-2A is
correctly folded and justifies the use of the isolated

It has been shown previously that the isolated CBM20
domain from AtPWD has relatively low affinity
towards cyclodextrins [20]. SPR was employed to mea-
sure the affinity of StGWD CBM45-2A for selected
soluble oligosaccharides. The domain was biotinylated,
immobilized on a streptavidin-coated chip and probed
for carbohydrate-binding ability at pH 8.0 (Fig. 3).
The resulting dissociation constants (K
d
) towards both
a- and b-cyclodextrin, as well as 6-O-a-maltosyl-b-
Fig. 2. Differential scanning calorimetry (DSC) analysis of
StGWD CBM45-2A. Reference subtracted thermograms of 50 l
M
StGWD CBM45-2A in 25 mM Hepes, pH 8.0. The full black line,
grey line and broken black line are the thermograms of the first,
second and third scans, respectively, at a rate of 1 °CÆmin
)1
.
Starch-binding domains in the CBM45 family M. A. Glaring et al.
1178 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS
cyclodextrin, were in the submillimolar range and com-
parable with the values obtained for the AtPWD
CBM20 (Table 1) [20]. The related function of the cat-
alytic modules in At PWD and StGWD is thus
matched by a similar range of affinity of their SBDs,
despite the fact that they have been assigned to two
different CBM families. This property was confirmed
by SPR analysis of the binding of b-cyclodextrin to
AtAMY3 in a similar experimental set-up, using a dif-

Although binding was evident in both cases, the heat
responses were small. The data obtained at pH 7.0
were noisy, suggesting that the protein was more prone
to aggregation at this pH. A one-site binding model
was fitted to the integrated ITC data, giving a K
d
value
of 0.68 ± 0.02 mm for the binding of b-cyclodextrin
to StGWD CBM45-2A at pH 8.0 (Fig. 4), in good
agreement with the value obtained using SPR
(Table 1). The measured heat of dilution was negligible
and was disregarded in the integrations. The binding
was driven by a favourable enthalpy change, which
compensated for an unfavourable entropy change
(DH = )42.1 ± 0.9 kJÆmol
)1
; TDS = )24.1 kJÆmol
)1
).
The binding affinity at pH 7.0 (K
d
= 0.44 mm) was
similar to that at pH 8.0. This thermodynamic finger-
print is consistent with the binding of b-cyclodextrin to
other SBDs [33].
The observed binding affinity of CBM45 for cyclo-
dextrins is considerably lower than that of other char-
acterized SBDs from microbial amylolytic enzymes.
Analysis of CBM20 and CBM21 SBDs from two glu-
coamylases gave K

, chi-squared test value for the fitted curve.
Protein Ligand K
d
(mM) R
max
(RU) v
2
(RU
2
)
StGWD CBM45-2A b-Cyclodextrin 0.38 ± 0.03 33 ± 0.61 0.29
a-Cyclodextrin 0.40 ± 0.06 36 ± 1.4 1.4
6-O-a-Maltosyl-b-cyclodextrin 0.44 ± 0.04 35 ± 0.97 0.51
AtAMY3 b-Cyclodextrin 0.19 ± 0.20 38 ± 0.88 1.3
AtPWD CBM20
a
b-Cyclodextrin, pH 7.0 1.07 ± 0.19
b-Cyclodextrin, pH 9.0 0.56 ± 0.12
a
Previously published data [20].
M. A. Glaring et al. Starch-binding domains in the CBM45 family
FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1179
Similarly, a CBM41 SBD from Thermotoga maritima
pullulanase showed a K
d
value of 42 lm for the inter-
action with b-cyclodextrin [35]. An amylase from
Bacillus halodurans carrying both a CBM25 and a
CBM26 SBD gave K
d

small avidity effect cannot be entirely ruled out.
AtAMY3 binding to starch in vitro
The full-length AtAMY3 offered an advantage when
examining the binding affinity of CBM45 SBDs to
starch, as it displayed catalytic activity and, being a
full-length enzyme, misinterpretation of binding data
as a result of instability or aggregation would most
likely be minimal compared with the isolated CBM45s.
Hence, the starch-binding ability of purified recombi-
nant AtAMY3 was demonstrated by incubation with
starch isolated from leaves of tobacco plants. Binding
was carried out at 4 °C and the a-amylase activity of
the unbound fraction was subsequently measured. A
one-site binding model was fitted to the binding iso-
therm (Fig. 5), resulting in a K
d
value of 36 ±
6.8 mgÆmL
)1
and maximum binding capacity (B
max
)of
93 ± 5.6%. This affinity is up to two orders of magni-
tude lower than that reported previously for the bind-
ing of various CBM20 domains to starch [6]. A similar
experiment using maize starch resulted in comparable
affinity, but substantially lower binding capacity
(K
d
= 21 ± 9.5 mgÆmL

The data obtained not only support the affinity
range measured using SPR and ITC with the cyclodex-
trin starch mimics, but also verify the starch-binding
ability of AtAMY3 in vitro. It cannot be precluded
that some binding may involve secondary binding sites
in the catalytic domain, but the demonstrated starch-
binding ability of different isolated CBM45s used in
both the present and other studies [7,10] suggests that
AtAMY3 does indeed interact with starch granules
through the tandem CBM45 domains.
CBM45 interaction with starch granules in planta
It has been shown by immunoblotting that StGWD
binds to starch in planta in its active full-length form
[23]. To investigate whether the isolated CBM45s from
StGWD function as SBDs in planta, they were C-termi-
nally fused to yellow fluorescent protein (YFP), either
singly or as the double module, and transiently
expressed in tobacco leaves. The constructs were tar-
geted to the chloroplasts by an N-terminal fusion to the
transit peptide of Arabidopsis GWD1. In a similar
experimental set-up, fusions between green fluorescent
protein (GFP) and either full-length AtAMY3 or the
tandem CBM45s were analysed. Investigation of locali-
zation using confocal laser scanning microscopy showed
clear targeting to the chloroplasts of mesophyll cells
and binding to disc-shaped transient starch granules
for StGWD CBM45-2, CBM45-1,2 and full-length
AtAMY3 (Fig. 6). The StGWD CBM45-1 fusion, in
contrast, gave rise to numerous highly fluorescent inclu-
sion body-like structures with no clear targeting (data

(A) StGWD CBM45-2 fused to YFP. (B) StGWD CBM45-1,2 fused
to YFP. (C) Full-length AtAMY3 fused to GFP. Scale bar, 20 lm.
Starch (mg·mL
–1
)
0 50 100 150 200
AtAMY3 bound (%)
0
20
40
60
80
100
Fig. 5. Binding of AtAMY3 to tobacco leaf starch in vitro. Recombi-
nant AtAMY3 protein was incubated with starch isolated from
leaves of Nicotiana benthamiana for 45 min at 4 °C. Unbound pro-
tein was assayed for activity by measuring the release of reducing-
end sugars from amylopectin. Each data point (±SE) is the average
of four independent experiments.
M. A. Glaring et al. Starch-binding domains in the CBM45 family
FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1181
were demonstrated to exhibit up to two orders of mag-
nitude lower affinity towards both cyclodextrins and
granular starch, compared with typical SBDs encoun-
tered in microbial amylolytic enzymes. This behaviour
is analogous to that of the CBM20-type SBD from
AtPWD [20] and supports the hypothesis that low-
affinity SBDs are important for dynamic and reversible
interactions in starch metabolism [28]. It remains
unclear how the functionality of these low-affinity

(Sigma-Aldrich). Following centrifugation, the supernatant
was loaded onto a HisTrap HP, 5 mL column (GE Health-
care, Uppsala, Sweden) and eluted by a 40–400 mm imidaz-
ole gradient in 20 mm Tris ⁄ HCl pH 8.0, 500 mm NaCl,
10% v ⁄ v glycerol and 0.5 m betaine according to the manu-
facturer’s instructions.
TEV protease cleavage of the His-tag was performed
with AcTEV protease according to the manufacturer’s
instructions (Invitrogen). For large-scale production, incu-
bation was performed overnight at room temperate using
25% of the recommended amount of protease. The cleaved
untagged protein was purified by anion exchange on a
Mono Q 10 ⁄ 100 GL column (GE Healthcare) in 20 mm
Tris ⁄ HCl pH 8.0 and eluted with 20 column volumes of a
0–0.5 m NaCl gradient. After dialysis to remove NaCl, the
protein was stored at 4 °C.
Cloning, expression and purification of
A. thaliana AMY3
An AtAMY3 cDNA clone (At1g69830, accession number
AY050398) was obtained from the RIKEN Arabidopsis
Genome Encyclopedia (RARGE, ).
Full-length AtAMY3 excluding the chloroplast transit pep-
tide and stop codon (amino acids 68–887) was amplified
(primers AtCBM1-NcoI and AtpAMY- NotI, Table S1) and
cloned into the NcoI and NotI sites of the expression vector
pET-28a containing a C-terminal 6 · His-tag. The con-
struct was transformed into E. coli BL21 Rosetta (DE3)
cells (Novagen). Protein expression was carried out in either
a 5 L bioreactor (Biostat B, B. Braun Biotech International,
Melsungen, Germany) on defined medium [37] by induction

approximately 1 mgÆmL
)1
and stored at 4 °C.
DSC analysis
DSC analysis was performed using a VP-DSC calorimeter
(MicroCal, Northampton, MA, USA) with a cell volume of
0.52061 mL at a scan rate of 1 °CÆmin
)1
. Samples were
dialysed in at least 500 volumes of 25 mm Hepes–NaOH,
pH 7.0 or pH 8.0, and degassed for 10 min at 20 °C. Base-
line scans collected with buffer in the reference and sample
cells were subtracted from sample scans. The reversibility of
the thermal transitions was evaluated by checking the
reproducibility of the scan on immediate cooling and
rescanning. The initial screening of the conformational sta-
bility of purified StGWD constructs was performed using a
Starch-binding domains in the CBM45 family M. A. Glaring et al.
1182 FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS
protein concentration of 0.5 mgÆmL
)1
in 25 mm Hepes–
NaOH, pH 8.0. The DSC analysis of the form with the
highest T
m
value (StGWD CBM45-2A) was performed fol-
lowing cleavage of the His-tag with TEV protease (see
above), and subsequent repurification and dialysis of 50 lm
protein as mentioned above. Origin v7.038 software with a
DSC add-on module was used for data analysis, T

range 0–2000 lm for each carbohydrate dissolved in the same
buffer. All data evaluation was carried out using the Biacore
T100 evaluation software.
ITC analysis
Experiments were performed using an MCS isothermal
titration calorimeter (MicroCal). Titrations were performed
by injecting 5 lL b-cyclodextrin in 25 mm Hepes–NaOH
pH 7.0 or 8.0 into a stirred (400 rpm) 1.3187 mL cell con-
taining 50 lm StGWD CBM45-2A in the same buffer. For
each titration of enzyme, the dialysis buffer of the sample
was titrated as a control using the same b-cyclodextrin
stock to measure the heat of dilution. The control titration
consisted of 10 injections of 1 lL in 2.5 s for the first injec-
tion and 5 lL for the rest, and with 180 s of equilibration
between injections. Titrations of the protein were carried
out similarly, but were continued until no significant
response was observed on ligand injections. Origin software
supplied with the instrument was used to analyse the data.
Starch-binding assays
Tobacco leaf starch was isolated from 5-week-old Nicoti-
ana benthamiana. The harvested leaves were homogenized
in 0.2% SDS in a polytron PT3000 blender (Kinematica
AG, Lucerne, Switzerland) and filtered sequentially through
2 · 100 lm and 2 · 20 lm filtration cloth. Following cen-
trifugation, the starch pellet was washed twice in 0.2%
SDS, three times in water, twice in 96% ethanol and air
dried.
Recombinant AtAMY3 (3 lg) was incubated with
tobacco leaf starch granules in a 350 lL mixture containing
20 mm Hepes pH 7.5, 0.5 mm CaCl

and cloned into the vector pPS48uYFP using an improved
USERÔ (uracil-specific excision reagent; New England
Biolabs, Ipswich, MA, USA) cloning procedure [39]. The
chloroplast transit peptide of Arabidopsis GWD1 (amino
acids 1–77) was fused to each construct by simultaneous
cloning of both fragments as described previously [10]. The
full-length ORF of AtAMY3 (primers AMY3-F and
AMY3-R, Table S1), as well as an N-terminal fragment
(amino acids 1–391) covering both CBM45s (primers
AMY3-F and AMY3SBD-R, Table S1), were fused to
enhanced GFP in the binary vector pK7FWG2 [40] using
GATEWAYÔ cloning technology (Invitrogen). Constructs
were transformed into Agrobacterium tumefaciens and tran-
siently expressed by infiltration in Nicotiana benthamiana as
described previously [41]. Expression and localization were
analysed by a confocal laser scanning microscope (TCS
SP2, Leica Microsystems, Wetzlar, Germany) equipped
with a 20 ·⁄0.70 or 63 ·⁄1.20 PL APO water immersion
objective. A 488 nm laser line was used for excitation, and
emission was detected between 520 and 550 nm for YFP
fluorescence, between 510 and 535 nm for GFP fluores-
cence, and between 600 and 750 nm for chlorophyll auto-
fluorescence.
M. A. Glaring et al. Starch-binding domains in the CBM45 family
FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1183
Acknowledgements
MAG was supported by a grant from the Danish
Research Council for Technology and Production Sci-
ences (grant no. 274-06-0312) and MJB by a Hans
Christian Ørsted postdoctoral fellowship from the

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