Received: 5 July 2018 Revised: 27 September 2018 Accepted: 16 October 2018

DOI: 10.1002/prot.25624

RESEARCH ARTICLE

Modulation of glucan-enzyme interactions by domain V in

GTF-SI from Streptococcus mutans
Manuel I. Osorio1,2 | Matías A. Zúñiga1,2 | Fernanda Mendoza2 | Gonzalo A. Jaña2 |
Verónica A. Jiménez2

1
Fisicoquímica Molecular, Universidad Andres
Bello, Santiago, Chile Abstract
2
Departamento de Ciencias Químicas, Glucansucrase GTF-SI from Streptococcus mutans is a multidomain enzyme that catalyzes the
Facultad de Ciencias Exactas, Universidad synthesis of glucan polymers. Domain V locates 100 Å from the catalytic site and is required
Andres Bello, Talcahuano, Chile
for an optimal activity. Nevertheless, the mechanism governing its functional role remains elu-
Correspondence
sive. In this work, homology modeling and molecular dynamics simulations were employed to
Verónica A. Jiménez and Gonzalo A. Jaña,
Departamento de Ciencias Químicas, examine the effect of domain V in the structure and glucan-binding ability of GTF-SI in full
Universidad Andres Bello, Sede Concepción, and truncated enzyme models. Our results showed that domain V increases the flexibility of
Autopista Concepción-Talcahuano 7100, the α40 -loop-α400 motif near the catalytic site resulting in a higher surface for glucan associa-
Talcahuano, Chile.
Emails: veronica.jimenez@unab.cl;
tion, and modulates the orientation of a growing oligosaccharide (N=8-23) in glucan-enzyme
gonzalo.jana@unab.cl complexes towards engaging in favorable contacts throughout the protein, whereas in the
Funding information truncated model the glucan protrudes randomly from domain B towards the solvent. These
Fondo Nacional de Desarrollo Científico y results are valuable to increase understanding about the functional role of domain V in GH70
Tecnológico, Grant/Award Number: 1150704
glucansucrases.

KEYWORDS

domain V, glucan-enzyme interactions, GTF-SI, molecular dynamics

1 | I N T RO D UC T I O N
Glucansucrases or glucosyltransferases are extracellular enzymes of
the glycoside hydrolase family GH70 that catalyze the synthesis of a
diversity of α-glucan polymers from sucrose such as dextran, mutan,
alternan, and reuteran, differing in the type of glycosidic linkage,
branching degree, length, and conformation.1,2 Glucansucrase GTF-SI
from Streptococcus mutans plays a major role in dental caries patho-
genesis, as it catalyzes the synthesis of the polymeric biofilm responsi-
ble for the formation of bacterial plaque on teeth, using sucrose as
glucose donor.3–5 Consequently, the inhibition of GTF-SI by active
molecules targeting this enzyme has emerged as an attractive strategy
to prevent tooth decay. The search for suitable inhibitors for GTF-SI
requires a detailed characterization of the structural and mechanistic
features responsible for glucan synthesis by this enzyme. The catalytic
mechanism of glucansucrase has been extensively discussed in the lit-
erature, and involves a two-step transglycosylation reaction, in which
a glycosyl-enzyme intermediate is formed after sucrose hydrolysis. In
This work was performed at Universidad Andres Bello, Chile.
the first reaction step, one glutamate residue acts as acid/base cata-
lyst, one aspartate behaves as nucleophile, and a second aspartate
moiety acts as a transition state stabilizer. The second reaction step
takes place at the same catalytic site and corresponds to a glucosyl
transfer to an acceptor, such as a growing glucan chain.1,2,6,7 The
resulting polymeric biofilm is a mixture of soluble and insoluble glu-
cans with mostly α-(1!3) linkages (85%) and a minor proportion of
α-(1!6) linkages (15%).6 This polymeric biofilm possesses high rigidity
and resistance thus enabling favorable conditions for bacteria prolifer-
ation on teeth.4
From a structural perspective, GTF-SI is a multidomain enzyme
with a common organization with members of the GH70 family. This
protein is composed of five major domains, namely A, B, C, IV, and
V. The catalytic core is formed by domains A, B, and C, which resem-
ble enzymes of the GH13 family, whereas domains IV and V are exclu-
sive for the GH70, and extend the overall protein structure.5,8
Experimental reports dealing with truncated enzymes have shown the
importance of domain V on providing an optimal activity for GTF-SI,
nevertheless the structural features governing its mechanism of action
have remained elusive, as the structure of GTF-SI has only been

Proteins. 2018;1–7. wileyonlinelibrary.com/journal/prot © 2018 Wiley Periodicals, Inc. 1

2 OSORIO ET AL.
solved in the absence of domain V, and released under the PDB code
5
3AIE. This partial structure can be employed to build a homology
model of the full enzyme based on the coordinates of a structurally
related protein containing domain V such as the crystal structure of
the N-terminal truncated GTF180 of Lactobacillus reuteri 180
(GTF180-ΔN) under PBD code 3HZ3.8 This model contains five dis-
tinctive linearly arranged domains, in which the polypeptide chain fol-
lows a U-path with each domain formed by two discontiguous
polypeptide chains from both the N- and C-terminal segments, except
for domain C (Figure 1). The 3HZ3 structure encompasses the com-
plete C-terminal segment of domain V but lacks the N-terminal vari-
able region composed of ~740 residues.8 The absence of this segment
has no implications in the validity of the 3HZ3 model to account for
the catalytic properties of GTF180, as truncation experiments have
shown that deleting the N-terminal variable segment does not affect
the catalytic activity of GTF180.9 On the other hand, the C-terminal
region on domain V has been proposed to participate in glucan-
enzyme association, thus constituting a relevant structural segment
for the model.6,10–12 Regarding domain V conformation, the crystal
structures of different glucansucrases revealed a large positional vari-
ability for this segment, while other domains remain essentially unal-
tered.13 The position of domain V shows a shift of about 20 Å in
GTFA-ΔN compared to GTF180-ΔN, whereas in ΔN123-GBD-CD2
of DSR-E this domain adopts a completely different position close to
the catalytic core, resulting in a more compact enzyme structure.14–16
The intrinsic flexibility of domain V has been related to the GTF180
functioning, as this domain may facilitate glucan synthesis by bringing
the polysaccharide chains toward and away from the catalytic site.5,13
Additionally, the flexibility of domain V in GTF180 has been related to
the ionic strength conditions under which crystallization takes place,
as observed in the case of L. reuteri N-terminally truncated glucansu-
crase GTF-180 in its orthorhombic apo-form (PDB code 4AYG), in
which high ionic strength conditions lead to a more curved conforma-
tion of domain V compared to the 3HZ3 model.14 Given that the fully
active enzyme corresponds to the 3HZ3 structure, this model was
employed as template to conduct our current molecular modeling
study.
FIGURE 1 Structure of GTF180-DN, GTF-SI, and the full model of GTF-SI containing domain V. The superposition of the active site residues
between GTF180-DN and GTF-SI model is displayed
In this work, a complete model of GTF-SI was built by homology
modeling using the crystal structure of GTF180-ΔN (PDB code 3HZ3)
as structural template. Constructing such model is valuable to gain
insight into the role of domain V on the structural and conformational
features of GTF-SI that could be relevant for the catalytic properties
of this enzyme. The full and truncated GTF-SI models were subjected
to fully atomistic molecular dynamics (MD) simulations to retrieve
atomistic information regarding the effect of domain V on the overall
protein motion and conformation. Additionally, MD simulations were
carried out to examine the role of domain V on glucan-enzyme inter-
actions considering α-(1!3) oligosaccharide chains with increasing
number of glucose units from N = 8 to N = 23. Our results reveal that
domain V strongly influences the flexibility of protein regions in the
proximity of the catalytic site, in terms of widening the glucan-binding
region on domain A. On the other hand, negligible changes were
observed in the structure and conformation of the catalytic site, thus
suggesting a minor influence of domain V on the intrinsic catalytic
parameters of GTF-SI. Analysis of glucan-enzyme complexes revealed
that domain V modulates the orientation of the growing oligosaccha-
ride chain toward enabling the interaction of the product with the
C-terminal glucan-binding segment of domain V, which has been pro-
posed in the literature as a region responsible for glucan association.14
These results are valuable to increase understanding about the factors
that determine GTF-SI activity and could be relevant for the design of
novel active inhibitors targeting this enzyme.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Systems preparation
The initial coordinates for the truncated GTF-SI enzyme were
retrieved from the structure released under the PDB code 3AIE, which
contains an octameric arrangement (chains A to H) of the polypeptide
chain (Uniprot sequence ID P13470, residues 244 to 1087) with a res-
olution of 2.10 Å.5 The complete model of GTF-SI containing the
domain V was built by homology modeling using the coordinates of
OSORIO ET AL.
GTF180-ΔN (PDB ID: 3HZ3) from L. reuteri as structural template in
the SWISS-MODEL server. The coordinates for the substrate
(sucrose) were retrieved from the GTF180-ΔN model, and were
employed to build initial models for the truncated and full GTF-SI
complexes with sucrose, which are referred as GTF and GTF-V models
from now on, respectively. These models were subjected to the MD
simulation protocol described in Section 2.2, and the resulting equili-
brated structures were employed to generate the coordinates of
glucan-enzyme models with oligosaccharide chains of increasing num-
ber of glucose units (N = 8 to N = 23) containing α-(1!3) glycosidic
linkages, which are referred as GTF-gN and GTF-V-gN models, where
N stands for the number of glucose units forming the glucan chain.
The initial coordinates of the first glucan chain (N = 8) were generated
using the LEaP program within the Ambertools 15 suite release, and
the GLYCAM06 force field parameters for glucose and glycosidic link-
ages.17 The initial coordinates for GTF-g8 and GTF-V-g8 systems
were obtained by aligning the glycosidic ring of the glucan reducing
end with the nonreducing end of the acceptor substrate. The resulting
glucan-enzyme complexes were subjected to the MD protocol
described in Section 2.2. The coordinates of the glucan in the complex
after equilibration were retrieved, and employed to add five glucose
units to its nonreducing end to generate the coordinates of a larger
glucan with N = 13. This extended glucan (N = 13) was employed to
build a second set of glucan-enzyme complexes (GTF-g13 and GTF-V-
g13), which were then submitted to MD simulations. This process was
repeated consecutively until glucan-enzyme complexes with N = 23
were obtained. This chain length enabled the interaction of the nonre-
ducing end of the glucan with domain V, and thus it was considered
appropriate to conduct the current study.
2.2 | MD simulations
The models GTF, GTF-V, and the series of GTF-gN and GTF-V-gN
complexes were modeled considering the protonation states of ioniz-
able residues at pH 6.5 as determined by the H++ web interface. 18
All
systems were solvated with TIP3P water molecules in a 15 Å water
box, and counterions were added to achieve electrical neutrality using
the LEaP program capabilities, as implemented in the Ambertools15
17
release. Protein structures were modeled using the ff14SB force
field, whereas glucans were described using the GLYCAM-06 force
field19 as implemented in the AMBER 16 software. In the case of GTF
and GTF-V, a standard MD protocol was followed, as detailed next:
(1) 1500 steepest descent minimization steps followed by 3500 conju-
gate gradient minimization steps were carried out for water molecules
relaxation using harmonic constraints of 10 kcal for the protein and
ligand; (2) the entire structure was then minimized considering 1500
steepest descent minimization steps and 6500 conjugate gradient
minimization steps; (3) minimized systems were progressively heated
to 300 K, with harmonic constraints of 10 kcal to the carbon skeleton
during 0.5 ns; (4) the system was then equilibrated for 0.5 ns main-
taining the constraints and then by 5 ns without restrictions at 300 K
in a canonical ensemble (NVT) ensemble; (5) finally a 200 ns unre-
strained isothermal-isobaric ensemble (NPT) production dynamics was
carried out at 300 K and 1 atm with a time step of 2 fs. In the case of
GTF-gN and GTF-V-gN complexes, minimization and equilibration
3
protocols were identical to those applied to GTF and GTF-V systems
but the production dynamics was carried out during 20 ns, which was
proved to be appropriate to obtain equilibrated structures of glucan-
enzyme complexes with N = 8, 13, 18, and 23. All calculations were
performed using Graphics Processing Unit (GPU)-enhanced AMBER16
implementations.
2.3 | Trajectory analysis
Mean square root of the deviation (RMSD) trajectory analysis for GTF
and GTF-V systems was carried out using the mdlovofit20 program.
This program makes a robust alignment of the structures by means of
low-order-value-optimization, identifying the mobile regions in each
frame, in order to iteratively obtain an optimized alignment. With this
program, a sample of 100 frames (200 ns) of each trajectory was ana-
lyzed, determining the RMSD of the carbon skeleton atoms and the
root mean square of the deviation of the fluctuations of each residue
(RMSF). RMSD and RMSF were calculated with respect to the initial
structures for GTF and GTF-V models.
3 | RESULTS AND DISCUSSION
Fully atomistic MD simulations were employed to examine the role of
domain V on the structure and conformational properties of the glu-
cansucrase GTF-SI from S. mutans, aimed at providing molecular-level
information concerning the relevance of this domain in the modula-
tion of the catalytic performance of this enzyme. To this purpose, our
first task was to obtain a full model of GTF-SI containing domain V,
using the coordinates of the related enzyme GTF180-DN, which
shares a ~53% of similarity. The homology model of GFT-SI including
domain V is displayed in Figure 1. In this structure, the domain V
adopts a linear organization with respect to the other domains, and
contains the first 54, and the last 110 residues of the polypeptide
chain. Superposition of the common domains (A, B, C, and IV)
between GTF180-ΔN and GTF-SI revealed a high level of similarity
with a global model quality estimation of 0.78, which indicates a high
reliability for the constructed model. Concerning the catalytic site,
GTF-SI and GTF180-ΔN enzymes show highly superimposable struc-
tures, with an average RMSD of 0.793 Å for the catalytic residues
Asp477, Asp588, Glu515, and Arg475 (Figure 1). Based on this evi-
dence, we built a full substrate-enzyme GTF-SI model (GTF-V) by
retrieving the coordinates of the substrate (sucrose) from the
GFT180-ΔN crystallographic model. The GTF-V model is expected to
account for the catalytically relevant structural features of the GTF-SI
enzyme, as it encompasses: (1) the catalytic core composed of
domains A, B, C; (2) the connecting domain IV; (3) and the C-terminal
segment of domain V with glucan-binding ability. As stated earlier, the
N-terminal variable region that is absent in the model is not relevant
for the purposes of the present study, as this polypeptide segment
does not interfere with GTF-SI activity.8
The complete substrate-enzyme models (GTF-V) and its counter-
part lacking domain V (GTF) were subjected to 200 ns MD simulations,
aimed at obtaining equilibrated systems to explore the role of domain
V on the conformational properties of GTF-SI that can be related to its
4 OSORIO ET AL.
enzymatic activity. Equilibration of the systems under study was veri-
fied by means of RMSD calculations, which revealed that both systems
became stable after 10 ns of MD runs (data provided as Supporting
Information). Consequently, trajectory analysis was carried out during
the last 150 ns of MD runs, as detailed in the following section.
3.1 | Role of domain V on the conformation and
overall motion of GTF-SI
As a first approach to deal with the role of domain V on the structure
of GTF-SI, we explored the local conformational variations of the pro-
tein upon the incorporation of domain V. Local flexibility changes in
the protein upon the incorporation of domain V were examined by
means of RMSF calculations on GTF and GTF-V models as displayed
in Figure 2. Our results revealed that domain V is a highly flexible pro-
tein region and its presence entails significant local conformational
changes in specific residues located in the N-terminal segment of
domains IV, B, and A. On the other hand, the flexibility of the protein
residues belonging to the catalytic site (Asp477, Arg475, Asp588, and
Glu515 according to 3AIE residue numbering) resulted in almost no
alteration upon the incorporation of domain V, thus indicating that
this protein region is not critical for determining the active conforma-
tion of the catalytic center. These results indicate that, despite the rel-
evance of domain V for the enzymatic performance of GTF-SI, the
presence of this domain should have a minor influence on the intrinsic
catalytic parameters of this enzyme, which can be examined using
reduced enzymatic models such as in previously reported Quantum
Mechanics/Molecular Mechanics (QM/MM) approaches.21,22
A different result was obtained for a protein region located in
the neighborhood of the catalytic site, namely the α40 -loop-α400
motif, which has been recognized as a relevant segment for GTF-SI
activity.5,23 This motif is composed of the protein residues 592-627
according to the amino acid numbering of GTF-SI in the PDB code
3AIE. RMSF calculations revealed that the α40 -loop-α400 motif
FIGURE 2 RMSF (Å) results for protein domains in GTF and GTF-V
models obtained from the last 150 ns of MD simulations [Color figure
can be viewed at wileyonlinelibrary.com]
presents a considerably higher mobility in the GTF-V model, thus
suggesting that domain V noticeably affects the conformation of this
protein region. Due to this conformational flexibility, the α40 -loop-
α400 motif exhibited an opener average structure in the GTF-V model,
which could induce an increase in accessible surface in the proximity
of the catalytic domain (Figure 3). To address this issue, the solvent
accessible surface area (SASA) in a spherical region of 10 Å of radius
from the glycosidic oxygen of the substrate was measured during
the equilibrated trajectories for GTF and GTF-V models. The histo-
grams corresponding to SASA distribution for each model are dis-
played in Figure 3, revealing a wider surface distribution for the
GTF-V model containing domain V. These results suggest that the
increased flexibility of α40 -loop-α400 contributes to generate a wider
accessible surface near the catalytic site, which could facilitate the
association of a growing glucan chain within the enzyme during the
catalytic process. To further explore the role of domain V on glucan-
enzyme interactions, we employed the equilibrated structures of
GTF and GTF-V systems to build a series of glucan-enzyme com-
plexes, considering glucan chains with increasing number of glucose
residues, as detailed next.
3.2 | Role of domain V on glucan-enzyme
interactions
MD simulations (20 ns) were performed to evaluate the role of domain
V on the modulation of glucan-enzyme interactions in GTF and GTF-V
systems. To this purpose, a series of glucan-enzyme models were built
using the equilibrated structures of the enzyme with and without
domain V, and a set of oligosaccharide chains with N = 8, 13, 18, and
23 glucose units. These systems were designed to reproduce the essen-
tial structural aspects of the glucan-enzyme association, arising from
the growth of the oligosaccharide chain. The largest system under
study corresponds to N = 23, which allows the interaction between the
end of the oligosaccharide chain and the glucan-binding regions of the
C-terminal segment of domain V. RMSD analysis of the series of GTF-
gN and GTF-V-gN models indicates that systems equilibration was
achieved during the first 5 ns of simulation. Consequently, trajectory
analysis was carried out during the last 15 ns of simulation. Representa-
tive equilibrated structures for the largest systems under study, namely
GTF-g23 and GTF-V-g23, are depicted in Figure 4. Equilibrated struc-
tures for the corresponding complexes with smaller glucan chains are
provided as Supporting Information. Regarding the series of GTF-gN
and GTF-V-gN models, our results reveal that domain V strongly modu-
lates the orientation of the growing glucan chain within glucan-enzyme
complexes. In the case of GTF-gN models, the oligosaccharide chain
protrudes outside domain B toward the solvent and interacts mainly
with protein residues belonging to this domain, namely Gln383,
Lys384, Leu434, Ala435, Tyr962, Lys967, Asn1052, Leu1054,
Gly1055, and Arg1056. Minor interactions are observed with protein
residues belonging to domain IV, namely Ser906, Phe907, and Asp909,
and no interactions were observed with protein residues belonging to
the α40 -loop-α400 motif. On the other hand, in the GTF-V-gN models,
the growing oligosaccharide chain engages in a series of intermolecular
contacts with the protein throughout domains A, B, IV, and V
(Figure 4). According to our findings, the overall orientation of the
OSORIO ET AL. 5

FIGURE 3 (Left) Comparison between the average conformation of the α40 -loop-α400 motif in GTF and GTF-V models obtained from the last
150 ns of MD simulations. (Right) Comparison between the solvent accessible surface area SASA (Å2) of the catalytic domain in in GTF and
GTF-V models [Color figure can be viewed at wileyonlinelibrary.com]

growing end of the glucan is modulated by the interaction with residues
Arg596, Gln592, Asp593, Tyr610, Val608, and Gly609 contained in the
0 00
α4 -loop-α4 motif, which were not observed in the case of the GTF-
gN models. The main modulating interactions are the following: (1) two
hydrogen bonds between the OH40 and OH60 of the first glucose unit
with the side chains of residues Gln592 (11% of occupancy) and
Asp593 (26% of occupancy); (2) a hydrogen bond between the OH60
group of the third glucose unit with the N H hydrogen and the C O
oxygen of the peptide bond of the Tyr610 residue (72% of occupancy).
0
Additionally, a strong hydrogen bond was observed between the OH6
of the tenth glucose with the C O oxygen of the Ala315 peptide bond
(IV domain), which is maintained at less than 2.1 Å during 99% of the
simulation. This residue is conserved in the GH70 family, which could
indicate the fundamental role of this region for the proper functioning
of the enzyme. It is worth to note that the flexibility of the α40 -loop-
α400 motif in glucan-enzyme complexes of the GTF-V-gN type is
reduced compared to the flexibility observed in the glucan-free system
GTF-V, thus revealing the importance of this structural moiety in the
association of a growing glucan chain during the catalytic process of
GTF-SI.
Concerning domain V, this protein region engages in a series of
intermolecular hydrogen bonds with the segment comprised
between the 17th and the 21st glucose unit within the oligosaccha-
ride chain. The most relevant interactions take place with residues
belonging to YG repeats located in the C-terminal and N-terminal
segments of domain V, which have been proposed to play a relevant
role in glucan-binding.24 These repeating units are composed of ~21
amino acids with a consensus sequence identified by the occurrence

FIGURE 4 Comparison between the average orientation of the growing oligosaccharide chain (N = 23) within glucan-enzyme complexes with the
GTF and the GTF-V models. Representative structures were retrieved from 20 ns molecular dynamics trajectories [Color figure can be viewed at
wileyonlinelibrary.com]
6 OSORIO ET AL.
FIGURE 5 Details about oligosaccharide-enzyme interactions with YG repeating motifs in domain V. Representative structures were retrieved
from 20 ns molecular dynamics trajectories [Color figure can be viewed at wileyonlinelibrary.com]
of a glycine three to five residues after a cluster of one to four aro-
matic residues including tyrosine.24 Examination of the amino acid
sequence revealed the existence of seven YG repeats in domain V,
two in the N-terminal segment, and five in the C-terminal segment;
four of them involved in relevant intermolecular contacts with the
oligosaccharide chain, as displayed in Figure 5. The first YG repeating
unit involved in glucan association is located at the N-terminal end of
domain V and comprises residues Tyr190 to Asn206. The most rele-
vant glucan-protein interactions involving this region are: (1) the
hydrogen bonds formed by the OH60 hydroxyl of the 20th unit of
the oligosaccharide with the side chain oxygen of Gln200, and the
carbonyl oxygen of the peptide bond of Lys199 with 40% and 20%
of occupancy, respectively; and (2) the hydrogen bond formed
between the side chain hydroxyl of Tyr190 and the oxygen OH20 of
the 21st glucose of the oligosaccharide chain with 25% of occu-
pancy. The remaining three YG motifs interacting with the oligosac-
charide chain are located in the C-terminal region of domain V and
comprise residues Asn1140-Asn1160, Gly1161-Tyr1180, and
Leu1181-Ala1195, respectively. The most relevant glucan-protein
interactions involving these regions are: (1) the hydrogen bond
between the side chain oxygen of Asn1155 and the O4’ of the 18th
glucose unit (38% of occupancy); (2) the hydrogen bond between the
0
peptide NH group of Asn1155 with the oxygen OH2 of the 19th
glucose unit; (3) the interaction between the side chain hydroxyl of
Tyr1167 and the oxygen OH60 of the 18th glucose unit (20% occu-
pancy); (4) the hydrogen bond formed between the side chain oxygen
of Asn1170 and the O6’ of the 17th glucose unit with 25% of occu-
pancy; (5) the interaction between the carbonyl oxygen from the
peptide bond of Leu1182 and the O6’ of the 18th glucose unit in
the glucan with 25% of occupancy; and (6) the interaction between
the carbonyl oxygen from the peptide bond of Gly1184 and the O6’
of the 18th glucose unit and with the O4’ of the 19th unit. The
extent of the interaction between domain V and the oligosaccharide
throughout the 17th to the 21st glucose units indicate that this pro-
tein domain is critical to maintain glucan-enzyme contacts during the
synthesis of the polysaccharide chain. According to this, the trun-
cated enzyme would have fewer regions of interaction with the
glucan and could be able to stabilize shorter polymer chains. On the
other hand, the full enzyme containing domain V would have a larger
glucan-binding area, thus enabling the stabilization of larger polymer
chains. These results are consistent with the experimental observa-
tions obtained with the complete and truncated GTF180 enzyme
(without the V domain), which revealed that truncation of the
enzyme impairs its polysaccharide-synthesizing ability.13,25 Our
results are also consistent with a processive enzymatic mechanisms
for GTF-SI, as it generates large-size glucans without releasing inter-
mediate products. Additionally, the identification of YG glucan-
binding repeats in domain V is a valuable result for our computational
approach as molecular-level information has been provided to account
for the role of domain V in the orientation of a growing oligosaccharide
chain in the GTF-SI enzyme.1,25,26
4 | CONC LU SIONS
MD simulations were carried out to examine the role of domain V on
the structural properties that could be relevant for GTF-SI activity.
Our results revealed that the incorporation of domain V noticeably
affects the local flexibility of protein regions that are relevant for glu-
can binding in domain A, such as the α40 -loop-α400 motif, which experi-
enced a larger mobility in the GTF-V model compared to the
truncated GTF system. The larger conformational freedom of the α40 -
loop-α400 motif induces an increase in the SASA in the proximity of
the catalytic site, which could enable a better interaction with growing
OSORIO ET AL.
oligosaccharide chains resulting from the catalytic process. On the
other hand, the conformation of the catalytic site on domain A
remained almost unaltered upon the incorporation of domain V, thus
suggesting that this domain has a minor influence on the intrinsic cat-
alytic parameters of the GTF-SI enzyme. Regarding glucan-enzyme
interactions, our results revealed that domain V exerts a strong modu-
lation of the orientation of the growing oligosaccharide chain within
the enzyme models in terms of enabling a higher number of glucan-
enzyme contacts throughout domains A, B, IV, and V, whereas in the
case of the truncated GTF model, the growing oligosaccharide pro-
trudes outside domain B toward the solvent with minimum contacts
with the protein outside this domain. To the best of our knowledge,
this is the first computational approach that has specifically addressed
the role of domain V as modulator of the glucan-enzyme interactions,
and this information could be valuable to enhance understanding
about glucansucrases of the GH70 family.
ACKNOWLEDGMENTS
Authors thank FONDECYT under grant number 1150704 for financial
support. M. I. O. thanks Doctorado en Fisicoquímica Molecular at Uni-
versidad Andres Bello for his doctoral fellowship.
ORCID
Verónica A. Jiménez https://orcid.org/0000-0002-6783-5657
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SUPPOR TI NG I NFORMATION
Additional supporting information may be found online in the Sup-
porting Information section at the end of the article.
How to cite this article: Osorio MI, Zúñiga MA, Mendoza F,
Jaña GA, Jiménez VA. Modulation of glucan-enzyme interac-
tions by domain V in GTF-SI from Streptococcus mutans. Pro-
teins. 2018;1–7. https://doi.org/10.1002/prot.25624