Biochem. J. (2008) 416, 27–36 (Printed in Great Britain)
27
doi:10.1042/BJ20080580
Crystal structures of the starch-binding domain from Rhizopus oryzae
glucoamylase reveal a polysaccharide-binding path
Jung-Yu TUNG*, Margaret Dah-Tsyr CHANG†, Wei-I CHOU†, Yen-Yi LIU*, Yi-Hung YEH*, Fan-Yu CHANG†, Shu-Chuan LIN†,
Zhen-Liang QIU* and Yuh-Ju SUN*1
*Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, and †Institute of Molecular and Cellular Biology, National Tsing Hua
University, Hsinchu 30013, Taiwan
GA (glucoamylase) hydrolyses starch and polysaccharides to β-Dglucose. RoGA (Rhizopus oryzae GA) consists of two functional
domains, an N-terminal SBD (starch-binding domain) and a
C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of
the SBD from RoGA (RoGACBM21) and the complexes with
β-cyclodextrin (SBD–βCD) and maltoheptaose (SBD–G7) were
determined. Two carbohydrate binding sites, I (Trp47 ) and II
(Tyr32 ), were resolved and their binding was co-operative. Besides
the hydrophobic interaction, two unique polyN loops compris-
ing consecutive asparagine residues also participate in the
sugar binding. A conformational change in Tyr32 was observed
between unliganded and liganded SBDs. To elucidate the
mechanism of polysaccharide binding, a number of mutants were
constructed and characterized by a quantitative binding isotherm
and Scatchard analysis. A possible binding path for long-chain
polysaccharides in RoGACBM21 was proposed.
INTRODUCTION
sequence identity, they indeed have similar biological function
and structural folding [16]. Therefore the most recent evolutionary
study on CBM20 and CBM21 proposes that these two SBD
families are grouped into a new CBM clan [17]. However, the
detailed binding mechanisms between CBM21 and starch/glycogen remain unclear.
SBDs can be functionally independent of the catalytic domains
and have been proposed to increase the hydrolysis of granular
starch by disrupting its structure and concentrating the catalytic
domains on the surface of starch [14,18–20]. Ligand binding
of RoGACBM21 with starch and soluble oligosaccharides was
determined by Chou et al. [15]. The K d values were of a similar
order of magnitude to that of CBM20 from AnGA (Aspergillus
niger GA), but the maximal amount of bound protein
(Bmax ) of RoGACBM21 was 40- to 70-fold higher than
that of AnGACBM20 [21,22]. The binding affinity between
RoGACBM21 and soluble ligands, βCD (β-cyclodextrin) and
G7 (maltoheptaose), was measured as approx. 5 μM [15].
Three-dimensional structures of several starch-binding CBM
superfamily members have been reported: CBM20 from AnGA
[23], CBM21 from RoGA [16] and Homo sapien phosphatase 1 (PDB: 2EEF), CBM25 and CBM26 from Bacillus halodurans maltohexaose-forming amylase [24], CBM34 from Thermoactinomyces vulgaris α-amylase [25], CBM41 from Klebsiella
aerogenes pullulanase [26] and CBM48 from Rattus norvegicus
AMP-activated protein kinase [27]. In the present study,
we determined the crystal structures of apo-SBD and the
complexes with βCD (SBD–βCD) and maltoheptaose (SBD–G7).
Additionally, a number of RoGACBM21 derivatives containing
point mutations in aromatic residues (Y32A, W47A, F58A,
Y67A, Y83A, Y93A and Y94A) and hydrophilic residues (N29A,
K34A, N50A, E68A, N96A and N101A) were isolated and
studied. We describe detailed interactions between RoGACBM21
and two substrates (βCD and G7) and propose a polysaccharidebinding path.
Starch, the primary source of stored energy in plants, is composed of amylose and amylopectin. The former consists almost
entirely of α-(1,4)-D-glucopyranose units; however, a few α1,6 branches and linked phosphate groups may be found [1,2].
The latter is composed of α-(1,4)-D-glucose segments connected
by approx. 5 % α-1,6 branching sites [3]. Both amylose and
amylopectin fold into helical structures [4,5] and are further
organized into the semicrystalline granular form. In the crystalline
state, amylose forms double helices [6], whereas in solution
it is a flexible chain that can adopt a locally helical shape.
In animals, glycogen, similar to the starch in plants, is the
storage form of glucose. Glycogen has structural similarities
to amylopectin, except that the number of branching sites and
glucose units are, in general, more than that of amylopectin.
GA (glucoamylase), also known as amyloglucosidase or γ amylase (EC 3.2.1.3), is a biocatalyst capable of hydrolysing
α-1,4 and α-1,6 glycosidic linkages in raw starches and related
oligosaccharides to produce β-D-glucose [7,8]. The GA from
Rhizopus oryzae (RoGA) comprises a C-terminal catalytic
domain, classified as a member of the GH15 (glycoside hydrolase
family 15) [9], connected to an N-terminal SBD (starch-binding
domain) by an O-glycosylated linker [10]. CAZY (http://www.
cazy.org/) classification, which is based on primary sequence
similarity, groups the SBD of RoGA into CBMs (carbohydratebinding modules) family 21 (RoGACBM21) [11,12].
To date, 51 CBM families have been classified, and eight of
them (families 20, 21, 25, 26, 34, 41, 45 and 48) have starchbinding activity [13]. The dissociation constants, K d , for the binding of SBDs to starch are in the micromolar range [14,15];
however, SBDs associated with GA only appear in two families
(CBM20 and CBM21). Previous reports on structure-based
molecular modelling and NMR spectroscopy of RoGACBM21
revealed that although these two families share very low
Key words: carbohydrate-binding module, β-cyclodextrin, maltoheptaose, Rhizopus oryzae glucoamylase, starch-binding domain.
Abbreviations used: AnGA, Aspergillus niger GA; CBM, carbohydrate-binding module; βCD, β-cyclodextrin; G7, maltoheptaose; GA, glucoamylase;
PEG, poly(ethylene) glycol; RMSD, root mean square deviation; Ro GA, Rhizopus oryzae GA; SBD, starch-binding domain.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
28
J.-Y. Tung and others
EXPERIMENTAL
Protein expression and purification
Recombinant enzyme expression, purification and the functional
assay for RoGACBM21 have been reported previously [15].
However, the quality and quantity of full-length RoGA protein
expression was poor due to the flexible O-glycosylated linker.
Briefly, the DNA fragment encoding RoGACBM21 was cloned
into the pET23a(+) expression vector and overexpressed in
Escherichia coli BL21-Gold (DE3) cells (Novagen). The recombinant SBD sample was purified by His-BindR affinity column
chromatography (Novagen). The purified RoGACBM21 sample
was dialysed against sodium acetate buffer (50 mM, pH 5.5). The
resulting C-terminally His6 -tagged SBD (11.65 kDa) was purified
by Ni-NTA (Ni2+ -nitrilotriacetate) affinity chromatography with
a final yield of 5 mg of purified protein per litre of cells.
Site-directed mutagenesis
All RoGACBM21 mutants were generated using the PCR-based
QuikChange® site-directed mutagenesis method (Stratagene) as
described [15] with pET-RoGACBM21 as the template, two
complementary primers containing the desired mutation, and
Pfu Turbo DNA polymerase (Stratagene). All constructs were
transformed into competent E. coli BL21-Gold (DE3) for protein
expression.
Quantitative measurement of binding to starch
The starch-binding isotherm was analysed by a saturation-binding assay as reported previously [15]. Wild-type and mutant
RoGACBM21 derivatives (100 μl, 5–50 μM) in sodium acetate
(50 mM, pH 5.5) were mixed with 0.1 mg of prewashed insoluble
starch and incubated at 25 ◦C with gentle stirring for 16 h.
After centrifugation at 16 000 g for 10 min at 4 ◦C, the protein
concentration of the supernatant (unbound protein) was determined by the BCA (bicinchoninic acid) protein assay, and the
amount of bound protein was calculated from the difference
between the initial and unbound protein concentrations. The K d
and Bmax values were determined by fitting to the non-linear
regression of the binding isotherms using a standard single-site
binding model.
Quantitative measurement of binding to soluble carbohydrate
Fluorescence spectrophotometry of the binding of wild-type
or mutant RoGACBM21 to βCD was recorded by measuring
changes in the intrinsic protein fluorescence intensity. Experiments were performed in 50 mM sodium acetate (pH 5.5) at
25 ◦C using a PerkinElmer LS-55 spectrophotometer. Circular and
linear carbohydrates (2–20 mM) were titrated into RoGACBM21
(1 μM, 2 ml), and the fluorescence-emission spectrum was
monitored at 350 nm with a fixed excitation at 280 nm. The
relative changes in fluorescence intensity were plotted against
the ligand concentration, and the data were fitted to a simulated
curve using the appropriate equation for a single binding site.
Crystallization
Crystallization trials were carried out using the hanging-drop
vapour-diffusion method. Both protein (1 μl) and reservoir (1 μl)
solutions were mixed and equilibrated against a reservoir solution
(500 μl) in Linbro plates. Initial crystallization conditions were
obtained using Hampton Research Crystal Screen kits and
then further optimized to obtain diffraction-quality crystals.
The concentration of RoGACBM21 used for crystallization was
c The Authors Journal compilation c 2008 Biochemical Society
Table 1 X-ray diffraction data and refinement statistics of apo-SBD and the
complexes of SBD–βCD and SBD–G7
Crystal
apo-SBD
SBD–βCD
SBD–G7
X-ray diffraction data
Wavelength (Å)
Resolution
Space group
Unit cell (a/b/c) (Å)
0.9732 (BL13C1)
1.25
P21 21 21
26.64/33.89/93.39
0.9762 (BL13C1)
1.8
P21 21 21
42.58/42.72/70.06
277182
23978
98.5 (93.9)
54409
12310
99.1 (94.1)
0.9998 (BL13C1)
2.3
P21
37.69/110.91/61.16
β = 90.72 ◦
76379
22511
99.4 (97.4)
31.5 (6.7)
41.3 (2.2)
14.3 (2.5)
Reflections collected
Unique reflections
Completeness (%):
overall (outmost shell)
I/σ (I): overall (outmost
shell)
R merge (%): overall
(outermost shell)∗
Refinement
Resolution (Å)
R (%)†
Rfree (%)‡
Used reflections
Residues
Atoms (non-hydrogen)
Sugar molecules
Atom of sugar molecule
Zinc atoms
Sulfate molecules
Water molecules
RMSD for bond length (Å)
RMSD for bond angle (o )
Average B-factors
All protein atoms (Å2 )
βCD (Å2 )
G7 (Å2 )
Zinc atom (Å2 )
Sulfate molecules (Å2 )
Water molecules (Å2 )
Overall (Å2 )
7.0 (29.4)
3.3 (44.5)
7.8 (39.4)
30.0–1.25
14.6
16.9
22701
106
836
–
–
–
–
210
0.01
1.38
30.0–1.8
22.4
23.8
10806
106
827
One βCD
77
1
–
126
0.007
1.3
30.0–2.3
23.6
27.4
18953
424
3,308
Four G7
312
–
6
316
0.009
1.5
9.31
–
–
–
–
23.00
12.06
29.28
32.25
–
32.90
–
42.27
31.10
13.46
–
19.53
–
30.08
23.07
14.8
∗
Rmerge = |Ih − <I >|/ Ih , where Ih is the measured intensity and <I > the average
intensity of reflection hkl .
†R = ||F obs |−|F calc ||/|Fobs |, where Fobs is the observed and Fcalc the calculated structure
factor of reflection hkl .
‡Rfree is calculated on the ‘test set’ of reflections.
approx. 10 mg/ml. The βCD and G7 were used at a molar ratio
of 1:2 (protein/carbohydrate) to form SBD–βCD and SBD–G7
complexes respectively. Three SBD crystals grew in different
conditions at 293 K. The apo-SBD crystal was crystallized in
25 % PEG [poly(ethylene) glycol] 4000, the SBD–βCD crystal
was grown using 18 % PEG 8000 and 0.2 M zinc acetate, and
the SBD–G7 crystals were grown in 30 % PEG 8000 and 0.6 M
ammonium sulfate.
X-ray data collection
The X-ray diffraction data were collected at beamline BL13C1
at the NSRRC (National Synchrotron Radiation Research Center,
Taiwan). The data were processed and scaled using the program
HKL2000 [28]. The apo-SBD, SBD–βCD and SBD–G7 crystals
diffracted to 1.25, 1.8 and 2.3 Å (1 Å = 0.1 nm) resolution
respectively. Both apo-SBD and the SBD–βCD crystals belong
to the orthorhombic P21 21 21 space group with one molecule per
asymmetric unit and the V M [29] was calculated as 1.81 Å3 · Da−1
and 2.64 Å3 · Da−1 respectively. The SBD–G7 crystal belongs to
the monoclinic P21 space group with four molecules per asymmetric unit and the V M [29] was calculated as 2.74 Å3 · Da−1 (Table 1).
Crystal structures of the starch-binding domain of R. oryzae glucoamylase
Figure 1
29
Structure-based multiple-sequence alignment of SBDs
The alignment of SBD from R. oryzae GA (Ro GACBM21: PDB code 2DJM) with SBDs from Mucor circinelloides (Mc CBM21), H. sapiens protein phosphatase 1 (Hs CBM21: PDB code 2EEF),
A. niger glucoamylase (An GACBM20: PDB code 1AC0), B . halodurans maltohexaose-forming amylase (Bh CBM25: PDB code 2C3X and Bh CBM26: PDB code 2C3G), T. vulgaris α-amylase
(TvAI CBM34: PDB code 1UH3), K. aerogenes pullulanase (Ka CBM41: PDB code 2FHF) and R. norvegicus AMP-activated protein kinase (Rn CBM48: PDB code 1Z0M). Secondary structural
elements of Ro GACBM21 are indicated above the alignment as arrows (β-strands). The locations of β-strands of other CBM families are underlined. The aromatic residues (bold and boxed) and
polar residues (bold and shaded) involved in ligand binding in Ro GACBM21 are marked. The corresponding residues in other CBM family members are marked similarly. The asparagine residues
of the polyN loops (Asn46 –X–Asn48 –Asn49 –Asn50 –X–Asn52 and Asn96 –Asn97 –Asn98 –X–X–Asn101 ) of Ro GACBM21 are labelled with ∗ and Tyr67 and Tyr93 are labelled with 䊉.
Structural determination and refinement
The crystal structures of RoGACBM21 were determined using
the unliganded solution structure RoGACBM21 (PDB: 2DJM)
[16] as a search model by molecular replacement. The molecular
replacement program MOLREP [30] was used for phase determination. Data between 8.0 and 4.0 Å and a Patterson radius
of 20 Å were used to calculate the rotation and translation
functions. A similar procedure was applied for the three structures
and significant rotation and translation solutions were obtained.
Structural model building was carried out using XTALVIEW [31],
and the structural refinement was performed by CNS [32] and
the CCP4 program suite [33]. The final statistics of refinement
for apo-SBD and the complexes, SBD–βCD and SBD–G7, are
summarized in Table 1. The co-ordinates of the RoGACBM21
structures have been deposited in the PDB as the accession numbers 2VQ4 (apo-SBD), 2V8L (SBD–βCD) and 2V8M (SBD–
G7).
RESULTS AND DISCUSSION
CBM folding topology
Several CBM superfamilies, including CBM20, CBM21,
CBM25, CBM26, CBM34, CBM41 and CBM48, contain an
SBD in either the N- or C-terminus [24,25,34–40]. These SBDs
reveal low sequence identity but a similar immunoglobulin-like
folding topology. RoGACBM21 contains 106 residues, which has
low sequence identity (< 15 %) with AnGACBM20, as well as
that of other SBD-containing CBM families. A structure-based
multiple-sequence alignment of SBDs from nine representative
CBM superfamilies is shown in Figure 1. The sequence alignment
was carried out based on the overall structural folding of eight
β strands of SBDs and the corresponding ligand-binding sites.
Two types of SBD topologies, type I and type II [15], show a
similar overall structure by switching the first and last β strands.
Superfamilies CBM20, CBM25, CBM26 and CBM41 belong to
the type I topology, whereas CBM21, CBM34 and CBM48 belong
to type II topology [15].
Overall structures
The RoGACBM21 and the SBD–βCD and SBD–G7 complexes
share a similar overall structure, which belongs to a typical βsandwich fold with an immunoglobulin-like architecture [20] with
approximate dimensions of 28 Å × 30 Å × 42 Å. The β-sandwich is symmetric and composed of eight β strands, which are
antiparallel except β7 and β8. These strands are paired as four
hairpin β strands, β12, β34, β45 and β67/8, and fold as a β
barrel. The β barrel contains a substantial hydrophobic core that
contributes the major stabilizing force to RoGACBM21. The
overall structures of SBD–βCD and SBD–G7 are shown (Figures 2A and 2B). Two carbohydrate-binding sites, designated as
sites I and II, were observed in both SBD–βCD and SBD–G7
complexes, in which the sugar ligands, βCD and G7, are located
at almost diagonal ends of the SBD in a perpendicular orientation.
Binding site I is located at Trp47 around loop β34 and site II is
located at Tyr32 around loop β23. The carbohydrate-binding ratio
for both SBD–βCD and SBD–G7 complexes is one sugar ligand
per SBD molecule in crystal, in which the SBD shares each
binding site with the symmetry-related molecule in the crystal
such that two SBD molecules together hold one sugar ligand.
Site I and site II are compatible to bind one sugar molecule cooperatively. In solution, the binding between the SBD and βCD is
determined to be 1:2 by isothermal titration calorimetry (results
not shown). The well-defined electron density map of βCD in the
SBD–βCD complex is shown in Figure 2(C). The K d (∼333 μM)
of the αCD complex is much higher than that of βCD and G7
complexes [15], because the cyclic ring of the six glucose units of
αCD is too small to fit the essential binding surface of the SBD,
such that its binding ability is dramatically reduced.
c The Authors Journal compilation c 2008 Biochemical Society
30
Figure 2
J.-Y. Tung and others
The overall structures of the Ro GACBM21 complexes
(A) A ribbon diagram of the SBD–βCD complex. Eight β strands are labelled as 1–8 and βCD molecules are shown in sticks and coloured in CPK. (B) A ribbon diagram of the SBD–G7 complex.
Maltoheptaoses are shown in sticks and coloured in CPK. (C) The 2|F o |−|F c | omit electron density map of βCD in the SBD–βCD complex. All direct binding residues in site I (Trp47 , Asn50 , Tyr83 ,
Asn96 , Asn101 and Tyr94 ) and site II (Tyr32 , Asn29 , Lys34 , Phe58 and Glu68 ) are shown. The omit map was contoured at 1.0 σ . (D) Superimposition of four maltoheptaoses of four molecules per
asymmetric unit in the Ro GACBM21–G7 complex. The key aromatic binding residues and maltoheptaoses are shown in sticks and coloured green, cyan, gold and yellow respectively. (E) Two polyN
loops in the SBD–βCD complex. The polyN loops (Asn50 and Asn101 ) are labelled and coloured in green and three aromatic sugar-binding residues, Trp47 , Tyr83 and Tyr94 are labelled. (F) Two polyN
loops of loop β34 (left-hand structure) and loop β78 (right-hand structure) in the SBD–βCD complex. Protein residues and βCD are coloured in green and CPK respectively. The hydrogen bonds
are indicated by broken lines.
There are four molecules per asymmetric unit in the SBD–G7
complex. The overall structures of four SBDs are very similar
with RMSD (root mean square deviation) values of 0.57–1.04 Å
in Cα. The key aromatic residues, Trp47 , Tyr83 , Tyr94 and Phe58
are in the same orientation, except for Tyr32 , which is still located
c The Authors Journal compilation c 2008 Biochemical Society
in the centre of G7 in the SBD–G7 complex, but tilted in a different
orientation (Figure 2D). The conformations of four G7s vary;
however, they tend to fold into a U shape and fit the curvature of
the SBD-binding site. The major conformational difference of G7
appears to be located in the two ends of molecules.
Crystal structures of the starch-binding domain of R. oryzae glucoamylase
Binding sites
47
Site I mainly comprises three conserved aromatic residues, Trp ,
Tyr83 and Tyr94 , to form a broad, flat and stable hydrophobic
environment. The aromatic rings of these residues construct the
curvature of the SBD-binding site and interact with the sugar
rings by ideal hydrophobic stacking interactions (Figure 2C).
Site I is rather rigid because Tyr83 and Tyr94 are located in β
strands and Trp47 is locked by Asn46 and Asn50 . Site II is generally
formed by loops β23 and β45, particularly dominated by two
aromatic residues, Tyr32 and Phe58 . Nevertheless, both Tyr32 and
Phe58 are in the proximity of the sugar ring where the van der
Waals surface and the βCD are closely packed. The sugar ring of
βCD is nearly parallel to the ring of Tyr32 , which protrudes into
the non-polar cavity of βCD and forms a hydrogen bond with
βCD O27 (Figure 2C). Such unique binding is only observed in
the SBD–βCD complex, where the α-glucan chains of βCD wrap
around Tyr32 to stabilize the binding. Similar binding patterns
were observed in CBM25, CD glycosyltransferase [41,42] and
CBM48, the glycogen-binding domain of AMP-activated protein
kinase [27], in which the corresponding residues in site II are
Leu600 and Leu146 respectively. Another key residue in site II is
Phe58 , of which the hydrophobic phenyl ring forms a flat stacking
interaction with the sugar ring of the glucosyl unit of βCD. Two
planar rings of Tyr32 and Phe58 pack closely against the van der
Waals surface of the βCD molecule and act like a clamp to pick
up the βCD.
In addition to the hydrophobic interactions, numerous
hydrophilic interactions between the SBD and βCD were
found. In site I, residues Asn50 (ND2-O61:2.7 Å), Asn96 (ND2O33:2.9 Å) and Asn101 (ND2-O22:2.6 Å; OD1-O32:2.9 Å) form
two unique polyN loops (Figure 2E) and provide the main
hydrophilic interactions to βCD. In site II, residues Asn29
(NH2-O3:2.8 Å), Lys34 (NZ-O3:2.8 Å and N-O3:3.3 Å) and Glu68
(OE1-O2:2.5 Å and OE2-O3:2.9 Å) supply several hydrogenbond interactions with βCD. Meanwhile, these residues interact
with each other by hydrogen bonds to provide a tight binding
environment for βCD and contribute the forces to stabilize the
SBD. The binding area of site II is small and narrow and it
protrudes away from the complex more than site I.
PolyN loop
Besides the hydrophobic interaction, several asparagine residues
from β34 and β78 near site I provide additional hydrophilic
interactions for the carbohydrate binding. These two unique polyN
loops positioned on the two sides of the sugar molecule interact
with the hydroxy groups to protect the hydrophobic curvature of
the binding-site groove created by Trp47 , Tyr83 and Tyr94 from
exposure to solvent (Figure 2E). The polyN loops consisting of
consecutive asparagine residues in loop β34 (Asn46 , Asn48 , Asn49
and Asn50 ) as well as in loop β78 (Asn96 , Asn97 , Asn98 and Asn101 )
(Figure 2F) participate in sugar binding.
The significant interaction networks created in each of the
polyN loops of both complexes stabilize the binding loop and
facilitate sugar binding. In polyN loop β34, residues Asn46
and Asn50 directly interact with the main chains of Trp47 such
that the aromatic ring of Trp47 forms hydrophobic stacking with
the sugar ring (Figure 2F). Meanwhile, Asn50 and Trp47 lock together to assist the sugar binding by a hydrogen bond (Figure 2F).
Furthermore, the geometry of Asn50 is squeezed out by
an interaction network, Asn46 –Trp47 –Asn48 –Asn50 –Gly51 –Asn52 –
Asn46 , and the side chain of Asn50 is forced to interact with the
sugar molecule (Figure 2F). Likewise, in polyN loop β78, Asn96
interacts with Ser99 , Ala100 and Asn101 by three hydrogen bonds
31
Table 2 Binding affinity of wild-type and mutant Ro GACBM21 for starch
and βCD
NB, no binding detected.
SBDs
Wild-type
Mutants of aromatic residues
Y32A
W47A
F58A
Y67A
Y83A
Y93A
Y94A
Mutants of hydrophilic residues
N29A
K34A
N50A
E68A
N96A
N101A
B max (μmole/g)
for starch
K d (μM)
for starch
K d (μM)
for βCD
41.1 +
− 1.1
1.4 +
− 0.1
5.1 +
− 0.7
23.4 +
− 0.7
23.0 +
− 1.3
37.5 +
− 1.4
3.7 +
− 0.1
6.4 +
− 0.1
27.8 +
− 0.2
27.0 +
− 1.1
0.8 +
− 0.1
4.4 +
− 0.6
4.6 +
− 0.5
6.5 +
− 0.4
7.1 +
− 0.4
2.7 +
− 0.1
3.5 +
− 0.5
6.0 +
− 0.7
NB
28.4 +
− 3.5
10.2 +
− 1.4
15.4 +
− 2.7
14.9 +
− 1.4
12.7 +
− 1.4
7.0 +
− 0.2
7.7 +
− 0.1
4.5 +
− 0.1
6.5 +
− 0.1
24.0 +
− 0.3
17.8 +
− 0.4
8.1 +
− 0.6
5.2 +
− 0.4
13.6 +
− 1.1
6.8 +
− 0.2
1.6 +
− 0.1
2.9 +
− 0.2
6.5 +
− 0.9
22.5 +
− 2.5
7.7 +
− 0.9
22.0 +
− 3.0
23.4 +
− 3.0
25.2 +
− 3.3
to build a βCD-binding network (Figure 2F). The orientation of
Asn101 is fixed by Asn96 and Asn97 with two hydrogen bonds,
as well as controlled by the adjacent residues, Ala100 , Tyr102 and
Gln103 . As a result, Asn101 binds βCD with two hydrogen bonds
(Figure 2F). A primary triangle interaction, Asn96 :CO–Asn101 :N–
Asn97 :NH2 , and a periphery interaction network, Asn96 –Ala100 –
Gln103 –Tyr102 –Gln95 –Asn97 , are produced and tight sugar binding
is generated. The orientations of Asn96 and Asn101 are especially
restricted and do not reveal favourable geometries in the
Ramachandran plot.
Some residues in the polyN loops show very restricted geometries because these residues have to link one by one and form
a series of chain interactions, which not only stabilize the binding
loops, but also confirm the sugar binding. These extra interactions from the polyN loops might produce a higher sugarbinding capacity and affinity for RoGACBM21 as compared with
those of other CBM superfamilies. Furthermore, the polyN loops
interact with two sides of the βCD molecule to assist in turning
the orientation of βCD to form hydrophobic interactions with
Trp47 , Tyr83 and Tyr94 . Interestingly, these characteristic polyN
sequences are only observed in RoCBM21 and McCBM21 (where
Mc is Mucor circinelloides) (Figure 1) [13,17].
Binding affinity for starch and βCD
To elucidate the mechanism of polysaccharide binding, we
examined the essential residues in the vicinity of the binding sites
according to the complex structures. A number of RoGACBM21
mutants were generated and characterized by a quantitative binding isotherm and fluorescence spectroscopic analysis (Table 2)
[15]. The mutant proteins contained substitutions at the aromatic
residues from site I (W47A, Y83A, and Y94A) and site II (Y32A
and F58A) involved in βCD and G7 binding and two additional
tyrosine residues near sites I and II, Y67A and Y93A. Mutations
at hydrophilic residues in site I (N50A, N96A and N101A) and
site II (N29A, K34A and E68A) involved in direct hydrogen-bond
interactions with ligands were also analysed.
The site I mutants (W47A, Y83A and Y94A) exhibited decreased binding affinity for starch and βCD when compared with
the K d values for wild-type RoGACBM21 (Table 2). The βCD
binding cannot be detected in the W47A mutant. Furthermore,
c The Authors Journal compilation c 2008 Biochemical Society
32
J.-Y. Tung and others
W47A, Y83A and Y94A showed a reduced Bmax for the binding
capacity to starch, especially Y83A, possibly because Tyr83 is
located on the inside position of the site I-binding pocket. Site I
(W47A) serves as the major carbohydrate-binding site for starch.
For the site II mutants (Y32A and F58A), the K d values of Y32A
for starch and βCD binding were similar to that of wild-type
SBD. In addition, Y32A had less effect on the binding of soluble
oligosaccharides. F58A showed an increased K d for the binding
of starch and had the highest K d for the binding of βCD among
all of the mutants; however, this mutant exhibited almost no effect
on the binding capacity for starch, with a Bmax similar to that of
wild-type SBD. Because Phe58 significantly affected the binding
to βCD and starch, Phe58 might play a key role in site II. The
Trp47 - and Tyr32 -binding sites were co-operative to sugar binding,
because the binding affinity of the single mutant, Y32A or W47A,
was partially maintained; however, the binding of the double
mutant (Y32A/W47A) to insoluble starch was almost completely
abolished [15]. The binding roles of Tyr32 and Trp47 can be
accommodated in RoGACBM21 complexes (Figure 2C).
From the structure-based alignment (Figure 1), two additional
interesting aromatic residues, Tyr67 and Tyr93 were studied. Tyr67
is conserved in most of the SBD-containing CBM superfamilies
and Tyr93 is also conserved in most of the CBM21 superfamily
members [13,17]. Although Tyr67 and Tyr93 do not directly interact
with sugar in both the SBD–βCD and SBD–G7 complexes
(Figure 2), Y67A and Y93A mutants showed apparently lower
Bmax and higher K d values (Table 2). In particular, Y67A had a
considerably reduced Bmax (3.7 μmol/g), the lowest starch-binding
capacity of all of the mutants. In the SBD–βCD complex, Tyr67
points into the N-terminal loop, in which the hydroxy group of
Tyr67 positions around Pro4 –Ser5 –Ser6 –Ala7 and forms a hydrogen
bond with the O atom of Ala7 and stacks with Pro4 in the N
terminus (Figure 3A). The mutant Y67A showed a small Bmax and
it might due to the instability of the N terminus. Thus Tyr67 might
play a role in the N-terminal stabilization. The overall structure of
Y67A should be maintained because the binding activity of Y67A
is preserved, and the secondary structure of Y67A examined by
circular dichroism was intact (results not shown). The result in
the present study demonstrates that Tyr67 might play an important
role in governing the starch-binding capacity for RoGACBM21.
The K d and Bmax values for mutants with substitutions at
hydrophilic residues in site I (N50A, N96A and N101A) and site
II (N29A, K34A and E68A) are shown in Table 2. Mutant N50A
had a reduced Bmax and the highest K d (∼ 10-fold higher than
wild-type) for starch binding among all site I and II mutants, and
this may be attributed to the loss of hydrogen-bond interactions
between Asn50 and Tyr83 . Asn50 might play a major role in the
binding of insoluble polysaccharides and contributes to the overall
integrity of site I. Mutants N96A and N101A (site I) as well as
K34A and E68A (site II) exhibited inefficient βCD binding but
with high K d values due to a loss of hydrogen-bond interactions.
Consequently, we suggest that these hydrophilic residues from
sites I and II might play a critical role in the binding of soluble or
insoluble polysaccharides, in either an individual or co-operative
manner.
A continuous polysaccharide-binding path
Based on sites I and II, as well as two crucial tyrosine residues
near sites I and II, Tyr67 and Tyr93 , a characteristic connection was
observed between two carbohydrate-binding sites (Figure 3A).
From the surface electrostatic potential distribution of the SBD–
βCD complex (Figure 3B), a continuous hydrophobic surface is
observed formed by residues Trp47 , Tyr83 , Tyr94 , Tyr93 and Tyr67 .
Furthermore, from the solvent-accessible surface of the SBD–
c The Authors Journal compilation c 2008 Biochemical Society
βCD complex (Figure 3C), there are two possible accessible
surface paths to link site I and site II. One path is through
residues Asn50 , Tyr83 , Tyr94 , Tyr93 and Tyr67 . From the mutation
data (Table 2), Y67A and Y93A mutants showed substantial
effects in the starch-binding assay, Tyr67 and Tyr93 might act
as a midpoint to link two binding sites and make a continuous
binding path for longer chain and larger polysaccharides, or even
starch. The distance of this potential polysaccharide-binding path
through Trp47 (site I), Tyr93 , Tyr67 and Tyr32 (site II) is in a range of
45–60 Å with an accessible surface area of approx. 1215 Å2 . The
other path is through hydrophilic residues, Asn50 , Lys85 , Glu87 ,
Lys35 and Lys34 , which are consecutive and it might be able to
link sites I and II also. The continuous binding path might be
important for the raw starch/insoluble polysaccharides, but not
for soluble and smaller carbohydrate molecules, such as βCD and
G7. During the initial binding step, sites I and II might act as two
persistent binding markers for soluble/insoluble polysaccharides,
after which the continuous polysaccharide-binding path may
assist further binding.
Liganded and unliganded Ro GACBM21
The superimposed structures of unliganded and liganded
RoGACBM21 are presented in Figure 3(D). They reveal
comparable overall structures, in which RMSD values between
the SBD and two complexes, SBD–βCD and SBD–G7, are 0.74
and 0.98 Å respectively, and the RMSD value between the two
complexes is 0.57 Å. The comparison of the residues around the
sugar-binding site of unliganded and liganded SBD is shown in
Figure 3(E). An apparent conformational change was observed
in Tyr32 , which formed a hydrogen bond with Asn29 in unliganded
SBD; however, the phenyl ring of Tyr32 flipped over and reoriented
to form sandwich stacking with the sugar ring upon ligand binding
(Figure 3E). Surprisingly, the essential aromatic residues (Trp47 ,
Tyr83 and Tyr94 ) as well as other ligand-binding residues remained
in the same orientation in unliganded and liganded SBDs. Before
ligand binding, numerous water molecules occupied the sugarbinding site and interacted with several hydrophilic residues, such
as Lys34 , Glu68 , Asn50 , Asn94 and Asn101 . However, upon ligand
binding, this water layer was replaced by a sugar molecule and
the interactions were established among the sugar and hydrophilic
binding residues.
These results indicated that the sugar-binding site of SBD is
rather fixed and stable. As long as the β barrel scaffold is formed
and the three key binding positions (Trp47 , Tyr83 and Tyr94 ) can
be preserved, the ligand binding is likely to take place. For linear
polysaccharides, the K d values of RoGACBM21 decreased with
increasing ligand length and it was worse than that of βCD [15].
The Y32A mutant significantly diminished the affinity in G7 but
had no effect in the short linear carbohydrates, such as maltotriose
and maltotetraose [15]. Tyr32 may adjust the orientation of its
phenyl ring to accommodate the conformation of a long ligand.
Ro GACBM21 complexes and other CBM superfamilies
The three-dimensional structures of two CBM21 members,
apo-RoGACBM21 and apo-HsCBM21 are superimposed in
Figure 4(A). They share a similar overall structure except for loops
β34, β45 and β56. The corresponding residues for ligand binding
residues in site I (Trp47 , Tyr83 and Tyr94 ) and site II (Tyr32 and Phe58 )
in the RoGACBM21 complex were also observed in HsCBM21
(where Hs is Homo sapiens) in putative site I (Trp72 , Ala114 and
Trp125 ) and site II (Phe59 and Tyr82 ) respectively. These residues
are located in comparable locations except that the orientations
Crystal structures of the starch-binding domain of R. oryzae glucoamylase
Figure 3
33
The polysaccharide-binding path
(A) A continuous polysaccharide-binding path in the SBD–βCD complex. Residues Trp47 (site I) and Tyr32 (site II) as well as Tyr67 and Tyr93 are shown as sticks and are coloured in red. βCDs
are shown as sticks and coloured in CPK. (B) The surface electrostatic potential of the SBD–βCD complex displayed using the program PyMOL (DeLano Scientific; http://pymol.sourceforge.net/)
with the negative potentials (red) and positive potentials (blue). Residues Trp47 , Tyr83 , Tyr94 (site I) and Tyr32 (site II) as well as Tyr67 and Tyr93 are labelled. (C) The solvent-accessible surface of
the SBD–βCD complex. The residues on the solvent-accessible surface were calculated by GPSS (http://gpss.mcsg.anl.gov/). Two possible polysaccharide-binding paths to link site I and site
II. Residues Trp47 , Tyr83 , Tyr94 , Tyr93 , Tyr67 and Tyr32 , as well as residues Asn50 , Lys85 , Glu87 , Lys35 and Lys34 are labelled. Residues Glu95 and Asn98 on the polyN loop β78 as well as residues
Ala7 and Gln10 around the N-terminus are labelled. (D) Structural superimposition of unliganded and liganded Ro GACBM21s. The structural differences are coloured as unliganded Ro GACBM21
(gold) and SBD–βCD (green) and SBD–G7 (cyan) complexes. Molecules βCD (green) and G7 (cyan) are coloured in complexes. (E) The sugar-binding site of unliganded (gold) and the liganded
Ro GACBM21s, SBD–βCD (green) and SBD–G7 (cyan). Molecules βCD (green) and G7 (cyan) are coloured in complexes. The key sugar-binding residues Asn29 , Tyr32 , Trp47 , Phe58 , Tyr83 and Tyr94
are drawn as sticks. The hydrogen bonds are indicated by broken lines.
of the side chains vary. We suggest that HsCBM21 might possess
binding characteristics similar to that of RoGACBM21.
AnGA, a GA from Aspergillus niger [23], shares the same
glycoside hydrolase characteristic as RoGA. However, the SBDs
of both proteins, AnGACBM20 and RoGACBM21, fold with
different topologies [15] and link with the catalytic domains
through their N- and C-terminal ends respectively [23,43].
The three-dimensional structures of the two SBDs could be
superimposed by switching the first and the last β strands; the
structural superimposition is presented in Figure 4(B). Most
CBM superfamilies contain one sugar-binding site; however,
RoGACBM21 and AnGACBM20 complexes contain two binding
sites [24–26,40]. RoGACBM21 binds co-operatively to sugar,
whereas AnGACBM20 exhibits independent sugar binding [22].
Even though the sugar-binding location is similar in both proteins,
several structural differences were noted (Figure 4B), particularly
c The Authors Journal compilation c 2008 Biochemical Society
34
Figure 4
J.-Y. Tung and others
Structural comparison of Ro GACBM21 with other CBM superfamilies
(A) Structural superimposition of unliganded Ro GACBM21 (PDB code: 2VQ4, green) and unliganded Hs CBM21 (PDB code: 2EEF, orange). Residues of site I (Trp47 , Tyr83 and Tyr94 ) and site II (Tyr32
and Phe58 ) in Ro GACBM21 and residues of site I (Trp72 , Ala114 and Trp125 ) and site II (Phe59 and Tyr82 ) in Hs CBM21 are shown as sticks and labelled. (B) Structural superimposition of Ro GACBM21
(PDB code: 2V8L, green) and An GACBM20 (PDB code: 1AC0, pink) complexes with βCD. Three main structural differences (loops β34, β45 and β78) are shown. The key residues (Trp47 , Tyr94
and Phe58 ) in Ro GACBM21 as well as the corresponding residues (Trp543 , Trp590 and Tyr556 ) in An GACBM20 are shown. To clearly examine the structural differences, the βCD molecules are not
shown. (C) Structural superimposition of Ro GACBM21 (PDB code: 2V8L, green) and Tv AICBM34 (PDB code: 1UH4, blue) complexes. The key residues of site I (Trp47 , Tyr83 and Tyr94 ) and site II
(Tyr32 and Phe58 ) in Ro GACBM21 as well as the corresponding residues of site I (Trp51 , Tyr89 and Tyr119 ) and site II (Trp65 and Trp77 ) in Tv AICBM34 are shown as sticks. The βCD (green) of the
Ro GACBM21 complex and the maltotridecaose (blue) of the Tv AICBM34 complex are shown as sticks.
in loops β34, β45 and β78 around the sugar-binding regions,
which are in completely different conformations. These distinct
loops provide the unique sugar binding for RoGACBM21
and AnGACBM20.
c The Authors Journal compilation c 2008 Biochemical Society
The two binding sites of AnGACBM20 and RoGACBM21
are structurally and functionally different and the corresponding
residues in AnGACBM20 are Trp543 and Trp590 for site I and Tyr527
and Tyr556 respectively. In AnGACBM20 [23], site I (Trp543 ) acts
Crystal structures of the starch-binding domain of R. oryzae glucoamylase
as the initial recognition site for starch, whereas site II (Tyr527 ) is
capable of recognizing a range of starch strand orientations. Site
I has a larger surface area, undergoes a conformational change
upon sugar binding, and is proposed to act as a more specific
site to lock the ligand into place. However, from the structural
results and functional assay data (Table 2) for the RoGACBM21
complexes, we assume that site I (Trp47 ) is the essential binding
site and site II (Tyr32 ) plays an auxiliary role as a recognition site
because a conformational change and the protruded orientation
of Tyr32 was clearly observed between liganded and unliganded
SBDs (Figure 3E). The residues corresponding to Trp47 in
most SBD superfamilies are highly conserved, except for
BhCBM26 (where Bh is B. halodurans) (Figure 1). Site I reveals
a larger and broader binding surface, and undergoes further
conformational changes upon βCD binding.
TvAI, an α-amylase from T. vulgaris [25] that catalyses the
hydrolysis of α-D-(1,4)-glucoside linkages in starch to release
the α-anomer, shows different hydrolase characteristics with
RoGA. However, both SBDs belong to the N-terminal starchbinding CBM superfamily and fold with the same type II topology
[25,43]. Although the sequence identity and similarity between
these two molecules are only 16 and 36 % respectively, their structures superimposed quite well, especially the β strands. The
structural superimposition of RoGACBM21 and TvAICBM34 is
shown in Figure 4(C) with an RMSD of 1.6 Å in Cα. Nevertheless,
structural differences were observed in loop regions, for example
loops β34 and β78 (Figure 4C). Two sugar-binding sites, siteNA and site-N, found in TvAICBM34 [25] correspond to site I
and site II respectively in RoGACBM21. Both sugar-binding
sites were in similar orientations but two sugar molecules were
in a perpendicular orientation in both proteins. Meanwhile, the
functional roles of the two sugar-binding sites in both proteins
were comparable: site I/site-NA binds sugar specifically and site
II/site-N helps the enzyme approach starch by recognizing the
starch surface.
There is a carbohydrate-binding curvature observed in several
CBM families, such as TvAICBM34 [25] and BhCBM25 [24]
complexes to provide the main protein–carbohydrate interactions
by several key residues, for example Trp51 , Tyr89 and Tyr119
in TvAICBM34 and His26 , Trp34 and Trp74 in BhCBM25. This
binding platform was also formed in RoGACBM21 by the
corresponding residues, Trp47 , Tyr83 and Tyr94 . These key residues
have a similar location in each protein structure, and their
aromatic rings hold the sugar molecules by hydrophobic stacking
interactions. Within this binding platform, the most conserved
residue is Trp47 in RoGACBM21, corresponding to Trp74 in
BhCBM25 and Tyr89 in TvAICBM34. This binding curvature
could swing around the substrate to create a tight binding pocket
and induce at least three glucose units of sugar molecules to be
bent in a segmented shape for binding.
Conclusions
The crystal structures of unliganded and liganded RoGACBM21
(SBD–βCD and SBD–G7) were determined at 1.25, 1.8 and 2.3 Å
resolutions respectively. Two carbohydrate-binding sites, I and
II, were found on the surface of SBD, of which site I is a flat
and broad hydrophobic-binding region created by the aromatic
residues Trp47 , Tyr83 and Tyr94 , and site II is a protruded and
narrow binding environment formed by Tyr32 and Phe58 . Site I
serves as the major sugar-binding site by forming an essential
binding curvature for at least three glucose units. Site II may play
an auxiliary role for recognizing the long-chain polysaccharides.
A significant interaction network, which was created in the
polyN loops unique to RoGACBM21, stabilizes the loops
35
and confirms the sugar binding. This might produce a higher
binding capacity for RoGACBM21 compared with other CBM
superfamilies. An especially stable binding environment in SBD
was suggested to accommodate a variety of sugars with different
conformations and lengths. From the structural determination and
the quantitative binding assay of RoGACBM21, a possible longchain insoluble polysaccharide-binding path was suggested, in
which the polysaccharide is bound by the specific binding sites I
and II as well as a continuous binding surface with Tyr67 and Tyr93
between two sites.
This work was supported by grants from the National Science Council of Taiwan (NSC
96-2311-B-007-014 and NTHU 96N2424E1 to Y.-J. S. and NSC 96-2627-B-007-003, to
M. D. T. C.). This work was partially supported by a Council of Agriculture grant, National
Science and Technology Programme for Agricultural Biotechnology (97AS-1.2.1.-STa5, to M. D. T. C.). The Ro GACBM21 clone was kindly provided by Mr Chia-Chin Sheu
(Simpson Biotech, Taoyan, Taiwan). The X-ray diffraction data were carried out at the
NSRRC (National Synchrotron Radiation Research Center, Taiwan).
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