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Simulation of the cavity-binding site of three bacterial

multicopper oxidases upon complex stabilization:
interactional profile and electron transference
pathways
a a b
Martiniano Bello , Jose Correa-Basurto & Enrique Rudiño-Piñera
a
Laboratorio de Modelado Molecular y Bioinformática de la Escuela Superior de Medicina ,
Instituto Politécnico Nacional México , Plan de San Luis y Diaz Mirón S/N, Col. Casco de
Santo Tomas, Mexico City , CP. 11340 , México
b
Departamento de Medicina Molecular y Bioprocesos , Instituto de Biotecnología,
Universidad Nacional Autónoma de México , Avenida Universidad 2001, Col. Chamilpa,
Cuernavaca , Morelos , CP 62210 , México
Published online: 16 Jul 2013.

To cite this article: Journal of Biomolecular Structure and Dynamics (2013): Simulation of the cavity-binding site of three
bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways, Journal
of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2013.817954

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 Journal of Biomolecular Structure and Dynamics, 2013
http://dx.doi.org/10.1080/07391102.2013.817954

Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex
stabilization: interactional profile and electron transference pathways
Martiniano Belloa*, Jose Correa-Basurtoa and Enrique Rudiño-Piñerab
a
Laboratorio de Modelado Molecular y Bioinformática de la Escuela Superior de Medicina, Instituto Politécnico Nacional México,
Plan de San Luis y Diaz Mirón S/N, Col. Casco de Santo Tomas, Mexico City, CP. 11340, México; bDepartamento de Medicina
Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001,
Col. Chamilpa, Cuernavaca, Morelos CP 62210, México

Communicated by Ramaswamy H. Sarma

(Received 16 May 2013; final version received 16 June 2013)
Downloaded by [University of Haifa Library] at 18:43 19 July 2013

Previous studies have shown that multicopper oxidases (MCOs) oxidize organic and inorganic compounds through
oxidation–reduction reactions in which three structurally and functionally arranged copper centers coordinate the uptake
of an electron from a reduced substrate. Structural comparisons among three bacterial MCOs, with high structural homol-
ogy and available three-dimensional information, reveal that the primary structural differences between these MCOs are
located near the mononuclear copper center (T1Cu), where substrate oxidation occurs, as opposed to where the reduction
of oxygen to water occurs at the trinuclear center. Nevertheless, this substrate oxidation is achieved through an outer-
sphere electron transfer mechanism that does not generate a stable substrate–enzyme complex. In this study, MCOs from
Thermus thermophilus (Tth-MCO), Bacillus subtilis (CotA), and Escherichia coli (CueO), which have been previously
determined through X-ray crystallography, were used as models to analyze the binding modes of these MCOs to three
organic molecules, with specific interest in the substrate-binding site. The binding mode of the electron-donor molecule
to the electron transfer binding site was primarily attributed to hydrophobic contacts, which likely play an important role
in the determination of substrate specificity. Some complexes generated in this study showed an electron donor molecule
conformation in which an electron could be directly transferred to the histidines coordinating T1Cu, while for others
additional electron transference pathways were also possible through the participation of charged residues during electron
transfer.
Keywords: multicopper oxidases; dynamic Simulations; transient complexes; electron transfer mechanism; protein–ligand
interactions

Introduction These copper centers coordinate the uptake of an

Multicopper oxidases (MCOs) are enzymes containing a
multiple copper center that catalyze the oxidation of a
broad range of substrates, including polyphenols,
substituted phenols, diamines, and inorganic compounds
(Kosman, 2010; Solomon, Sundaram, & Machonkin,
1996), concomitantly with the four-electron reduction of
O2 to H2O (Quintanar et al., 2007; Solomon, Augustine,
& Yoon, 2008). MCOs play roles in diverse physiologi-
cal processes, such as lignin synthesis and degradation
(Askwith et al., 1994; Hakulinen et al., 2002; Kosman,
2010; Messerschmidt et al., 1992; Roberts et al., 2002),
pathogenesis (Dick, Torpey, Beveridge, & Tebo, 2008),
and morphogenesis (Mayer & Staples, 2002).
The copper ions present in MCOs are structurally
and functionally arranged into three copper centers.
electron from a reduced substrate. First, the one-electron
oxidation of the substrate occurs in the vicinity of mono-
nuclear copper center type 1 (T1Cu) or the blue copper
site, whose tight coordination to cysteine generates an
intense adsorption band of approximately 600 nm, giving
the enzyme a blue color. The oxidation is coupled to the
four-electron reduction of O2 to H2O at a trinuclear cop-
per center (TNC) comprising copper ion type 2 (T2Cu)
and the binuclear copper ion type 3 (T3Cu) Solomon
et al., 2008.
The co-crystallized complex between the fungal
laccase Trametes versicolor and 2,5-xylidine was the
first MCO complex with organic compounds identified
(Protein Data Bank (PDB) entry 1KYA). The inspection
of this complex shows that the electron transfer cavity

*Corresponding author. Email: mbellor@ipn.mx

Ó 2013 Taylor & Francis

 2 M. Bello et al.
is rather wide, facilitating binding to larger substrate
molecules (Bertrand et al., 2002). This complex is
primarily stabilized through hydrophobic interactions
and two polar residues, H458 and D206, and the two
residues establish hydrogen bonds with the amino group
of 2,5-xylidine. Based on this information, Bertrand
et al. suggested that H458 could act as the primary
acceptor of electrons from the substrate, whereas D206
is conserved in fungal laccases (Piontek, Antorini, &
Choinowski, 2002). Furthermore, comparing the
substrate-binding sites between different fungal laccases
revealed several similarities (Zhukhlistova, Zhukova,
Lyashenko, Zaitsev, & Mikhailov, 2008). To date,
several crystallographic structures of MCOs have been
determined and are available in the PDB: ascorbate
oxidase (Messerschmidt et al., 1992), ceruloplasmin
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(Bento, Peixoto, & Zaitsev, 2007), fungal laccases
(Antorini et al., 2002; De la Mora et al., 2012; Ducros
et al., 2001; Ferraroni et al., 2012; Ferraroni et al.,
2007; Garavaglia et al., 2004; Hakulinen et al., 2002;
Kallio et al., 2011; Lyashenko et al., 2006; Polyakov
et al., 2009), Fet3p (Taylor, Stoj, Ziegler, Kosman, &
Hart, 2005), CueO (Roberts et al., 2002), CotA (Engui-
ta, Martins, Henriques, & Carrondo, 2003), phenoxazi-
none synthase (Smith, Camara-Artigas, Wang, Allen, &
Francisco, 2006), Tth-MCO (Serrano-Posada, Valderra-
ma, Stojanoff, & Rudiño-Piñera, 2011), and MCOc
(Silva et al., 2012). According to the three-dimensional
structures of MCOs (Antorini et al., 2002; Bento et al.,
2007; De la Mora et al., 2012; Ducros et al., 2001;
Enguita et al., 2003; Ferraroni et al., 2012; Ferraroni
et al., 2007; Garavaglia et al., 2004; Hakulinen et al.,
2002; Kallio et al., 2011; Lyashenko et al., 2006;
Messerschmidt et al., 1992; Polyakov et al., 2009;
Serrano-Posada et al., 2011; Silva et al., 2012; Smith
et al., 2006; Taylor et al., 2005), the structural topology
of these MCOs consists of three cupredoxin domains
connected by linker peptides, where the T1Cu is
localized in the third domain, while the TNC is located
between the first and the third domain (Murphy, Lind-
ley, & Adman, 1997). Furthermore, in our previous
work, we used molecular dynamics (MD) simulations
to show that proper copper coordination is essential to
observe the conformational mobility coupled to the
biologically active form, with all the copper sites occu-
pied (Bello, Valderrama, Serrano-Posada, & Rudiño-Piñ-
era, 2012). Based on the three-dimensional information,
all MCOs present a well-defined hydrophobic binding
site pocket of variable depths near T1Cu (Bertrand
et al., 2002; Enguita et al., 2004).
The available co-crystallized complexes have helped
to characterize the nature of the electron transfer
binding site (ETBS) and the key residues that stabilize
the complex transient formation. However, the chemical
nature of these complexes is not classical, and the
catalysis for MCOs occurs through an outer-sphere
electron transfer mechanism, in which the substrate and
the enzyme do not necessarily establish stable chemical
interactions (Marcus & Sutin, 1985). Therefore, this
characteristic could be responsible for the few
substrate-MCO complexes available in the PDB. In fact,
as far as we know, the CotA-2,2-azino-bis(3-ethylbenzo-
thiazoline-6-sulfonic acid (ABTS) complex is the only
bacterial MCO substrate complex for which the crystal-
lographic structure has been determined (Enguita et al.,
2004). For this complex, it was difficult to define the
precise details of adduct binding at a resolution of
2.4 Å (Enguita et al., 2004), and the poor quality of its
electron density made it difficult to determine the actual
incorporation of ABTS into the crystal (Bello et al.,
2012). A comparison of the substrate-binding sites
between the complexes of the fungal laccase from T.
versicolor bound to 2,5-xylidine and CotA-ABTS,
revealed differences with respect to the ligand confor-
mation in the ETBS, primarily reflecting substantial
differences in the shape and dimension of both ligands
(Zhukhlistova et al., 2008). Furthermore, the aromatic
system of the 2,5-xylidine in the crystal structure of the
laccase from T. versicolor is virtually perpendicular to
residue H458, and the imidazole ring of residue H457
in the CotA protein structure is nearly perpendicular to
the triazole ring of ABTS.
Consequently, MD simulations have been used to
characterize transient complex formation (Bello et al.,
2012) and attempt to improve the substrate specificity
toward a specific ligand (Gupta & Farinas, 2010). Never-
theless, more information is necessary to understand the
flexibility and dynamics of the MCOs’ binding sites and
identify the keys residues that stabilize the transient
complex. To address this problem, in this work, we per-
formed long MD simulations (0.1 μ) for each bacterial
MCOs, as well as modified forms of CueO (CueOm) and
Tth-MCO (Tth-MCOm). From which, 50 ns were
performed for the copper-coordinated formations (Tth-
MCO, CueO, CotA CueOm, and Tth-MCOm) and another
50 ns for the bound species, to evaluate the molecular
mechanisms and identify the key residues involved in
transient complex stabilization, and the energetic and the
dynamical properties relevant for the formation of the
electron transfer complex under aqueous conditions. The
three bacterial MCOs examined in this study share a
high degree of structural homology and adequate experi-
mental evidence exists to support the oxidizing activity
of these molecules toward three common organic com-
pounds: 2,6-dimethoxy phenol (3DM), syringaldazine
(SGZ), and ABTS Bello et al., 2012; Gupta & Farinas,
2010; Gupta, Lee, & Farinas, 2010; Kataoka et al.,
2007; Messerschmidt & Oxidases, 1997. However, in
contrast to most of the MCOs, the substrate-binding site
of CueO is deeply buried under a methionine-rich helical
 Cavity-binding site of three bacterial multicopper oxidases
region comprising α-helices 5, 6, and 7, which inhibits
the access of potential substrates (Kataoka et al., 2007;
Singh, Grass, Rensing, & Montfort, 2004). Interestingly,
the activity of these compounds, although at low levels,
toward ABTS, SGZ, and 3DM has been shown
(Kataoka et al., 2007; Singh et al., 2004). Our results
indicate that, for most of the generated complexes, a
similar enzyme–substrate complex is formed, in which
hydrophobic forces play a central role. In addition, the
electron donators for some complexes were located at a
distance to potentially donate an electron to one of the
two coordinating histamines of CuT1. In other cases,
additional pathways are possible that involve direct
electron transfer or involve charged residues that
coordinate the uptake and subsequent transfer of an
electron to CuT1.
Downloaded by [University of Haifa Library] at 18:43 19 July 2013
Methods
The crystal structures of the hyperthermophilic MCO
from T. thermophilus (Tth-MCO, PDB entry 2XU9) and
the MCO from B. subtilis (CotA, PDB entry 1UVW, this
PDB entry has recently been replaced (Enguita et al.,
2003) new PDB entry 3ZDW) were used; however, the
structural analysis between both entries does not show
significant changes (RMSD = 0.13 Å), and the interac-
tions between the ligand and the ETBS are similar (sup-
plementary material, fig. 1S). The MCO from E. coli
(CueO, PDB entry 1N68) and the mutant form of CueO,
for which the α-helix 4 has been deleted (CueOm, PDB
entry 2YXV), was used to build our MD models. A
mutant form of Tth-MCO (Tth-MCOm) was created by
deleting the linker connecting the β-strands 21 and 24 of
the second domain (loop (β21-β24)D2) residues 287–
312). The complexes between the Tth-MCO, CotA,
CueO, CueOm, Tth-MCOm, and the organic compounds
ABTS, SGZ, and 3DM were built by taking out the last
conformation obtained from the 50 ns-long MD simula-
tion of the copper-coordinated forms above depicted and
docking them with each one of the organic molecules in
the ETBS to create the following theoretical complexes:
Tth-MCO-SGZ, Tth-MCO-3DM, CotA-SGZ, CotA-
3DM, CueO-ABTS, CueO-SGZ, and CueO-3DM for
which another 50 ns-long MD simulation was run. Crys-
tallographic information was only available for the com-
plex CotA-ABTS (PDB entry 3ZDW has superseded
entry 1UVW since December 2012); therefore, it was
not needed to construct the theoretical complex, and the
electron density deposited for the ABTS moiety was
weak and noncontinuous. The coordinates for the equili-
brated copper-coordinated form of Tth-MCO (Holo) and
the Tth-MCO-ABTS complex were obtained from our
previous MD simulation study (Bello et al., 2012). For
the mutant species, only the CueOm-ABTS and Tth-
MCOm-ABTS complexes were constructed to determine
3
whether deleting these loops affects the ligand stabiliza-
tion. These two MCOs were chosen because they had
been reported for their the inability to obtain crystallized
complexes between both MCOs and organic compounds,
which have been attributed to the loop (β21-β24) and
α-helix 4 for Tth-MCO and CueO, respectively, two
protein regions that occlude the ETBS (Bello et al.,
2012; Kataoka et al., 2007). Furthermore, although for
CueOm an increment in activity toward organic com-
pounds, revealing that the lack of α-helix 4 favored the
access of organic compounds (Kataoka et al., 2007) has
been experimentally demonstrated, structural information
about the complex form is not available. Therefore, this
contribution could aid in designing a mutant for
Tth-MCO (Tth-MCOm), with which it would be possible
to obtain a crystallized complex. But for CueOm, whose
tridimensional structure is available, our study will con-
tribute to get insights into the map of interactions
involved in increasing the activity of CueOm with respect
to Cueo and based on this information to propose new
mutants. The coordinates for the amino acid residues that
had no density in the electron density map were
generated using the CPHmodels-3.0 server (Nielsen,
Lundegaard, Lund, & Petersen, 2010). PROCHECK
(Laskowski, MacArthur, Moss, & Thornton, 1993) was
used to validate the stereochemistry of the resulting
three-dimensional models (supplementary material
(Table 1S)).
All the MD simulations were performed in the
presence of explicit water molecules and Na+ counteri-
ons using GROMACS 4.0.5 software, with the
GROMOS 53A6 force field (Oostenbrink, Villa, Mark,
& van Gunsteren, 2004). The simulation protocol used
for our system was identical to that described in our
previous study (Bello et al., 2012). Through the
production runs, the trajectory data were saved every
1 ps, and the duration of the simulations was 50 ns for
holo forms and 50 ns for each complex form, summing
0.1 μ for each system. For each system, the analysis
was performed after reaching equilibrium. Several
geometrical properties, such as the root-mean-square
deviation (RMSD), root-mean-square fluctuation
(RMSF), hydrogen bonding, radius of gyration (RG),
and apolar, and polar solvent accessible surface areas
(SASA), were calculated according to our previous
studies (Bello et al., 2012). Average structures were
determined for the complex forms over the last 30 ns of
a 50 ns-long simulation, using the GROMACS program
g_covar. Energy calculations were obtained through
short Lennard-Jones (LJ), and Coulomb energy (Coul)
calculations were performed using the g_energy
module. The analysis was calculated using GROMACS
tools and homemade software. All the graphical
representations were obtained using PYMOL version
0.99rc6 (DeLano, 2002).
 4 M. Bello et al.
Results and discussion
Stability and geometrical properties
Tth-MCO-SGZ, Tth-MCO-3DM, Tth-MCOm-ABTS,
CotA-ABTS, CotA-SGZ, CotA-3DM, CueO-ABTS,
CueO-SGZ, CueO-3DM, and CueOm-ABTS formed
stable protein–ligand complexes. Before the ligand
reached a stabilized conformation in the ETBS, a differ-
ent region around the electron transfer site was scanned
for several ns. For almost all systems, the ligand reached
a stable conformation inside the binding site for the last
30 ns of a 50 ns-long simulation. Furthermore, through
that period of simulation, the RMSD oscillated approxi-
mately 0.18–0.41 nm (Table 1). The comparison of the
plots between the apo and holo forms (data not shown)
indicates that in general all the apo forms required less
time to reach equilibrium (15 ns), whereas the complex
forms reached equilibrium within the first 20 ns.
Downloaded by [University of Haifa Library] at 18:43 19 July 2013
Interactional analysis and average properties
The case of Tth-MCO
Tth-MCO is one of the few MCOs with available
three-dimensional information concerning its copper-
coordinated (holo) and free (apo) forms. Therefore, this
system has been used in studies characterizing the asso-
ciation between copper and protein mobility (Bello et al.,
2012). These studies revealed that the presence or
absence of copper coordination affects the conforma-
tional mobility of the loop (β21-β24) Bello et al., 2012.
However, crystallographic data for complexes between
Tth-MCO and organic compounds have not been
obtained, reflecting difficulties in obtaining the catalytic
nature of MCOs. Thus, MD simulations provide a
remarkable method to study these transient complexes
and have been used in the present study to characterize

However, apo forms were characterized by slightly
higher RMSD values, which suggests that despite com-
plex forms reaching equilibrium in longer simulation
times, under equilibrium times the complex forms were
less flexible than the apo forms, revealing that the ligand
confers more stability to the protein structure. The only
exception was observed for Tth-MCOm and Tth-MCOm-
ABTS where a slight RMSD value characterized the
complex form. Table 1 also shows the RG, SASA, and
hydrogen bonds (HB). Overall, these results are consis-
tent with the nanosecond time scale and show that the
copper-coordinated (Tth-MCO, Tth-MCOm CotA, CueO,
and CueOm) and bound states each protein did not show
significant differences, were stable during the MD simu-
lations and remained near the starting crystallographic
coordinates. Taken together, these parameters show that
the systems did not exhibit any significant conforma-
tional changes during the simulations.
the potential binding modes and important residues
involved in this complex formation using the open
conformation of Tth-MCO (Bello et al., 2012). MD
simulation revealed that ABTS is stabilized through
hydrophobic, hydrophilic, and charged residues, but the
major forces that drive the complex arrangement are
hydrophobic in nature (Bello et al., 2012). Furthermore,
although the thiazoline group is not located near the
H450 residue, a charged residue, D390, could participate
in electron transfer to T1Cu, as proposed for fungal
lacasses (Zhukhlistova et al., 2008). However, in the
present study, we explored the ligand-binding mode of
SGZ or 3DM in the ETBS (see methods) of Tth-MCO
because the catalytic activity of both organic compounds
has been previously reported (Bello et al., 2012; Mess-
erschmidt & Oxidases, 1997). Furthermore, to determine
whether this enzyme could conserve substrate affinity,
although a part of the substrate transfer site was not

Table 1. Geometrical properties of the free copper-coordinated and bound states.

System HBintra RG (nm) Apolar SASA (nm2) Polar SASA (nm2) RMSD equilibrium (nm)
⁄
Tth-MCO 202 ± 2 2.15 ± 0.01 192 ± 3 133 ± 2 0.41 ± 0.01
Tth-MCO-ABTS⁄ 204 ± 4 2.15 ± 0.01 195 ± 4 136 ± 3 0.19 ± 0.01
Tth-MCO-SGZ 202 ± 4 2.13 ± 0.01 190 ± 3 136 ± 2 0.19 ± 0.01
Tth-MCO-3DM 201 ± 5 2.14 ± 0.01 188 ± 3 136 ± 4 0.18 ± 0.01
Tth-MCOm 192 ± 4 2.1 ± 0.01 183 ± 4 127 ± 3 0.27 ± 0.01
Tth-MCOm-ABTS 192 ± 4 2.1 ± 0.01 185 ± 4 124 ± 2 0.33 ± 0.02
CotA 200 ± 11 2.3 ± 0.01 213 ± 3 165 ± 3 0.34 ± 0.01
CotA-ABTS 200 ± 11 2.3 ± 0.01 223 ± 4 172 ± 2 0.24 ± 0.01
CotA-SGZ 200 ± 4 2.3 ± 0.01 215 ± 4 166 ± 2 0.18 ± 0.01
CotA-3DM 201 ± 4 2.3 ± 0.01 215 ± 4 165 ± 3 0.18 ± 0.01
CueO 149 ± 4 2.2 ± 0.01 187 ± 4 150 ± 3 0.34 ± 0.01
CueO-ABTS 147 ± 4 2.18 ± 0.01 188 ± 4 152 ± 2 0.30 ± 0.014
CueO-3DM 147 ± 4 2.2 ± 0.01 188 ± 3 152 ± 2 0.32 ± 0.020
CueO-SGZ 149 ± 4 2.17 ± 0.01 186 ± 4 149 ± 3 0.25 ± 0.010
CueOm 142 ± 4 2.1 ± 0.01 176 ± 4 132 ± 3 0.30 ± 0.01
CueOm-ABTS 142 ± 4 2.0 ± 0.2 177 ± 3 132 ± 2 0.26 ± 0.015
⁄
These data were obtained from Bello et al. (2012).
 Cavity-binding site of three bacterial multicopper oxidases 5

present, we created a variant form of Tth-MCO, where
the loop (β21-β24) was deleted (see methods).
In the Tth-MCO-SGZ complex, SGZ was docked in
a similar position as observed for ABTS in the
Tth-MCO-ABTS complex (Bello et al., 2012). In the
substrate electron transfer site, the SGZ reached a
constant conformation after 18 ns of MD simulation.
Subsequently, until the end of MD simulation, the com-
plex was maintained primarily through hydrophobic
interactions (Table 2) between the second and third
domains (Figure 1(A) and (B)). This ligand conformation
placed the donor group at a distance of 6 Å from H450
(Figure 5(A)). Furthermore, D390 and D392 were
located near the electron-donor molecule and could
participate in the uptake of electrons from the electron-
donor molecule (Figure 1(B)).
Downloaded by [University of Haifa Library] at 18:43 19 July 2013
tion after 4 ns. Figure 4(C) and (D) show the average
conformation and detailed interactions for the
Tth-MCOm-ABTS complex, respectively. This complex is
stabilized through polar and charged residues, but
charged residues primarily drive the arrangement of the
complex. Several contacts observed for Tth-MCO-ABTS
(Bello et al., 2012) are lost and the ligand conformation
is changed, placing the electron donor group at a distance
of 9 Å from T1Cu (Figure 5(E)). However, D390 and
D392, which could participate in electron uptake, are
located in close proximity to the ABTS (Figure 4(D)), as
observed for Tth-MCO-ABTS. Thus, despite the trunca-
tion of loop (β21-β24), Tth-MCOm maintained its binding
properties.
The case of CotA

The Tth-MCO-3DM complex showed two constant

conformations for the 3DM molecule. The ligand
reached a stable conformation during the first 4 ns of
simulation, and this conformation was maintained until
20 ns of MD simulation (conformation 3DM1). Subse-
quently, that ligand conformation was lost and another
conformation (3DM2) was present at a more distant loca-
tion (Figure 1(C) and (D)). Interestingly, both conforma-
tions were located in hydrophobic regions, the second
more than the first (Table 2), and shared almost the same
distance and number of histidines that coordinate T1Cu
(Figure 5(A)). Furthermore, despite these distances,
3DM1 was located 7.4 Å from D392 (residue shown as
orange stick), sequential in sequence to H393, which
coordinates T1Cu. In contrast, 3DM2 is approximately
11 Å from D392, in a ligand conformation not geometri-
cally optimized to donate an electron to D392. Moreover,
although 3DM shows a constant conformation in both
depicted sites, this ligand shows higher mobility com-
pared with SGZ and ABTS (Bello et al., 2012), suggest-
ing that the ETBS in Tth-MCO facilitates bulky ligands.
The Tth-MCOm-ABTS complex shows that ABTS,
which was docked in a similar manner as in the
Tth-MCO-ABTS complex, reached a constant conforma-
Although diverse number of substrates oxidized through
CotA have not been identified, the oxidation of ABTS
and SGZ through MCO has been well documented
(Gupta et al., 2010). Both, the substrate-free and ABTS-
bound crystal structure are available, and the ABTS-
bound structure is nearly identical to the native enzyme
(Enguita et al., 2004; Enguita et al., 2003), suggesting
that no significant conformational changes occur in the
bound state. The bound structure shows that 18 amino
acids (at a distance criterion of <5 Å) surround the sub-
strate, and most of these residues are hydrophobic in nat-
ure (Enguita et al., 2004; Enguita et al., 2003). However,
the residues that stabilize the complex have not been
defined (Enguita et al., 2004). Therefore, we performed a
structural analysis of the MD simulations (50 ns) for the
complexes CotA-ABTS, CotA-SGZ, and CotA-3DM.
Our results showed that within the first 5 ns of MD sim-
ulation, the ligand scanned the ETBS for approximately
5 ns and subsequently reached a stable conformation,
establishing contacts into 5 Å with 14 residues (Table 2).
Figure 2(A) and (B) show that the average complex
between CotA and ABTS is stabilized through polar,
apolar, and charged residues, but mainly through apolar

Table 2. List of residues at 5 Å from each ligand, with a residence time of up to 2 ns.

Complex Residue
Tth-MCO-SGZ M195, N196, N229, A230, Y232, R290, P265, M354, M391, D390, D392, P394
Tth-MCO-3DM-1 Y232, R234, L262, P287, Y288, D289
Tth-MCO-3DM-2 R410, P411, F412, Y414, L252, A247, V404, V407, V421
Tth-MCOm-ABTS R231, Y232, A230, E199, G197, N196, D392, D390, M195, M354
CotA-ABTS P226, A227, F228, C229, C322, G323, G324, P384, L386, T415, R416, G417, T418, H419
CotA-SGZ C229, C322, G323, G230, A227, F228, P226, P212, E213, N214, L219, G382, P384
CotA-3DM P296, E298, T418, T466, I467, Q468, A469, I494
CueO-ABTS L298, V300, G378, MET379, H465, R466
CueOm-ABTS P274, S301, E476, N458, K460
CueO-3 dm S243, P274, L276, M277, V300, M385, G386, H387, M388, L442, K474
CueO-SGZ S243, V256, L264, V268, V270, L273, P274, L276, F281, S301, H381, L442, N458, R466, K474, E476
 6 M. Bello et al.
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Figure 1. Average conformations and detailed interactions between Tth-MCO and two different compounds. Complex Tth-MCO-
SGZ (A and B) and Tth-MCO-3DM1 and 3DM2, (C and D). The residues in close proximity (0.5 nm) to the two ligands (blue
spheres or stick models) are represented as green stick models. Although at a distance below 0.5 nm, CuT1 (blue sphere) and its
coordinating histidines are shown in Figures 1–4 to show its proximity with respect to the ligand.

residues. One half of the ligand was stabilized through
the loops of the second domain, and the other half of the
ligand was stabilized through residues in the third
domain, in close proximity to T1Cu. The sulfonate
moiety approached the disulfide bridge between C229
and C322, as reported for the co-crystallized complex
(Enguita et al., 2003). The remaining sulfonate group
was stabilized through several hydrophobic residues and
R416 (Figure 2(B)). This geometry places the thiazoline
ring near H497 (Figures 2(B) and 5(B)), favoring the
direct transfer of an electron from the electron-donor
molecule to T1Cu, subsequently transferring the electron
to the TNC through C492.
In the CotA-SGZ complex, SGZ, which was docked
in the electron transfer cavity in a similar conformation
as that observed in the CotA-ABTS complex, loses its
initial ligand conformation within the first ns of MD sim-
ulation, and experienced different ligand conformations
in the ETBS for approximately 7 ns. Subsequently, and
until the end of the MD simulation period, the electron-
donor molecule reached a stable conformation, establish-
ing contacts into 5 Å with 13 residues (Table 2). Figure 2
(C) and (D) show that SGZ is stabilized in an almost
linear conformation, similar to that observed for the
CotA-ABTS complex. Indeed, one half of SGZ is stabi-
lized through similar residues as those that coordinate
the CotA-ABTS complex (Figure 2(C) and (D)),
although with a different ligand–enzyme contact, reflect-
ing the chemical differences between both substrates.
The other 2,6 dimethoxyphenol group interacts with a
different set of apolar residues than those observed for
the CotA-ABTS complex. This latter interaction places
 Cavity-binding site of three bacterial multicopper oxidases 7
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Figure 2. Average conformations and detailed interactions between CotA and three different compounds. Complexes CotA-ABTS
(A and B), CotA-SGZ (C and D), and CotA-3DM (E and F). The residues in close proximity (0.5 nm) to the three ligands (blue
spheres or stick models) are represented as green stick models.

the donor group at a further distance than those from the
ABTS (Figure 5(B)). Because of this conformation, the
SGZ donor group was approximately 11 Å distant from
H497 (Figure 5(B)). However, the two charged residues,
E496 and D499, are localized at 6 and 8 Å from SGZ
(residues shown as orange stick, Figure 2(D)),
respectively, and are both sequential in sequence with
H497, which coordinates T1Cu. Therefore, this confor-
mation could lower the substrate specificity reported for
SGZ with respect to ABTS (Gupta & Farinas, 2010;
Gupta et al., 2010).
In the CotA-3DM complex, we observed that 3DM,
situated in a structural position similar to that of the
CotA-ABTS and CotA-SGZ complexes, lost its initial
conformation after the first 2 ns, then reached a stable
conformation surrounded by a group of residues totally
different from those identified for the complexes
CotA-ABTS and CotA-SGZ, in which the hydrophobic
 8 M. Bello et al.
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Figure 3. Average conformations and detailed interactions between Cueo and three different compounds. Complexes Cueo-ABTS
(A and B), Cueo-SGZ (C and D), and Cueo-3DM (E and F). The residues in close proximity (0.5 nm) to the three ligands (blue
spheres or stick models) are represented as green stick models.

ligand section established hydrophobic interactions with
several hydrophobic residues (Table 2), whereas the polar
atom of 3DM formed a hydrogen bond with that of A469
(Figure 2(F)). Interestingly, a similar behavior as that
described for CotA-SGZ was observed for the CotA-3DM
complex where, despite the donor group of 3DM being
placed at a distance (9 Å) that did not allow to establish a
direct electron transference to H497 (Figure 5(B)), a
charged group (E496) was localized at 7 Å from 3DM,
and sequential in sequence with H497 (residue shown as
orange stick, Figure 2(F)), which could participle in the
electron uptake and subsequent transfer to T1Cu.
 Cavity-binding site of three bacterial multicopper oxidases
The case of CueO
CueO oxidizes a wide variety of substrates, including
3DM (Kataoka et al., 2007; Roberts et al., 2002).
Intriguingly, little oxidase activity occurs in the absence
of excess copper (Kataoka et al., 2007). There is
adequate three-dimensional information concerning the
free state of CueO, despite sharing a high degree of
structural homology with CotA and Tth-MCO (Bello
et al., 2012). CueO possesses a methionine-rich helix
(residues 355–371, in which seven methionines are pres-
ent) and loop (residues 372–379, in which two methio-
nines are present) that lies over the T1Cu site and a
peptide region, which connects the end of this helix to
H404 (residues 380–403, five methionines) near the C-
terminal of this helix and the T1Cu center (Kataoka
et al., 2007). However, only methionines 303, 355, 364,
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368, 376, 379, and 385 from the CueO insert, and
methionines 440 and 441 from the neighboring β-strand,
form a seven-methionine cluster at the entrance to the
T1Cu site, which might be important for copper-depen-
dent activity (Roberts et al., 2002). Although, this region
could function as a barrier to the access of bulky organic
substrates (Roberts et al., 2002), the binding of copper to
these methionine residues could lead to the ordering of
this loop and the formation of a substrate-specific bind-
ing site (Roberts et al., 2002).
For most MCOs, the imidazole edge of one of the
histidines coordinating T1Cu (Tth-MCO (H450), CotA
(H497), CueO (H505)), which is solvent-exposed and in
close distance from the substrate-binding pocket, functions
as an effective pathway for electron transfer between the
substrate and T1Cu (Bertrand et al., 2002; Messerschmidt
et al., 1992; Zhukhlistova et al., 2008). However, for
CueO, α-helix 4 occludes this amino acid (H443) from the
solvent, avoiding direct interaction with organic substrates
(Roberts et al., 2002). Based on the crystallography data,
D439 could be the corresponding amino acid residue
involved in electron transfer to T1Cu. However, this
notion was discarded because of the side chain of D439
hydrogen bonds with H443, which remains in a native and
mutant form (Kataoka et al., 2007). However, the trun-
cated mutant also demonstrated an increment in activity
toward ABTS and the newly identified activity for SGZ,
indicating that the truncation favored the access of these
organic substrates to the region near the T1Cu site (Kat-
aoka et al., 2007). Nevertheless, because of the lack of
structural information for the co-crystallized complexes,
we performed MD simulations to analyze the residues
involved in the complex stabilization between the native
form of CueO, the three organic compounds showing
substrate activity, CueO-ABTS, CueO-SGZ, and CueO-
3DM (see methods), and CueOm bound only to ABTS
(CotAm-ABTS).
9
Our results showed that in the CueO-ABTS complex,
ABTS, which was docked in a small substrate cavity
under α-helix 4, lost this ligand conformation after the
first 1 ns and then the ABTS reached a conformation that
was maintained for the rest of the MD simulation
(Figure 3(A)). Figure 3(B) shows the residues involved
in complex stabilization, where one half of ABTS is
coordinated through a neutral (H465) and a charged
residue (R466), the middle of ABTS is coordinated
through two hydrophobic residues, and another region of
the sulfonate moiety forms interactions with M379
(Table 2). In this ligand conformation, the thiazoline ring
of ABTS is approximately 19 Å from H505 (Figure 5
(C)). Interestingly, this latter residue forms part of the
cluster of methionines likely involved in the putative
Cu+/Cu2+ traffic exchange and electron transfer to T1Cu.
For the CueO-SGZ complex, for which SGZ was
docked in a similar manner as in the CueO-ABTS
complex, SGZ explored the small substrate cavity under
α-helix 4, reaching a stable conformation after 10 ns.
Figure 3(C) and (D) show the binding conformation for
the CueO-SGZ complex and the residues involved in
complex stabilization, respectively. As shown in Figure 3
(D), the number of residues that stabilize the complex is
important (Table 2), and most of these residues are
hydrophobic in nature. However, charged residues are
also involved in complex stabilization, creating favorable
electrostatic potential to facilitate complex formation.
Although, this ligand conformation places the donor
group at approximately 12 Å from H505 (Figure 5(C)), a
charged residue (E476) is located near the donor group
of SGZ (Figure 3(D)), which could participate in the
uptake and subsequent transfer of an electron to T1Cu.
Furthermore, although there are two charged residue in
close proximity to T1Cu (E476 and D439), based on
crystallography evidence, D439 has been discarded to
participate in electron transference because its side chain
forms hydrogen bonds with H443, which remains in a
native and mutant form (Kataoka et al., 2007).
For CueO-3DM, 3DM scanned the small substrate
cavity under α-helix 4 of CueO for 15 ns and subse-
quently achieved a constant conformation (Figure 3(E)
and (F)). Figure 3(F) shows that 3DM was stabilized
through several hydrophobic and one charged residue
(K474). Indeed, K474 forms a hydrogen bond with the
methoxy group, whereas the donor group hydrogen
bonds with polar backbone atoms of L276. The donor
group was located approximately 13 Å from H505 (Fig-
ure 5(C)), and although SGZ is constructed by two 3DM
groups, this ligand was placed at a distance further (7 Å)
from E476 (residue shown as orange stick) than that
observed for SGZ (Figure 3(D) and (F)), suggesting that
a bulky group is more likely to enhance complex stabil-
ization. Furthermore, 3DM was also located near two
 10 M. Bello et al.
methionines, which could participate in the copper coor-
dination and electron transfer to T1Cu (Figure 3(F)).
For the CueOm-ABTS complex, the ligand explored
the ETBS for approximately 20 ns and subsequently
reached a constant conformation for the remainder of the
MD simulation. Figure 4(A) and (B) show the complex
conformation and the detailed interactions that stabilize
the complex, respectively. Figure 4(B) shows that one
half of the ligand is coordinated through two charged
residues, as observed for the CueO-ABTS complex, the
middle region is coordinated through a hydrophobic resi-
due and the other half interacts with a polar residue. The
ligand conformation places both thiazoline groups at an
average distance of 16.5 Å from H505 (Figure 5(D)).
Despite the deletion of α-helix 4, a group of methionines
remain, which could participate in copper coordination
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and potential electron transfer to T1Cu through E476,
although direct contacts with ABTS (>5 Å) were not
established (Figure 4(B)), and these interaction might
underlie the experimentally observed incremental activity
toward ABTS (Kataoka et al., 2007).
Figure 4. Average conformations and detailed interactions between the mutant forms of Tth-MCOm or Cueo and ABTS. Complexes
Cueom-ABTS (A and B) and Tth-MCOm-ABTS (C and D). The residues in close proximity (0.5 nm) to the ligand (blue spheres or
stick models) are represented as green stick models.
Energetics of complex stabilization
Table 3 shows the evolution of the interaction energies
between the ligands and the rest of the protein during
equilibrium (20–50 ns). Although the energy values were
unstable upon initiating the MD simulations, these values
reached equilibrium at 20 ns for almost all complexes.
Interestingly, for most ligands, the total energy
interaction was dominated through the favorable LJ inter-
actions, whereas the electrostatic contributions, although
favorable, slightly contributed to complex stabilization.
However, the opposite effect was observed for
Tth-MCOm-ABTS, CueO-ABTS, and CueOm-ABTS, in
which the electrostatic interactions were the major contri-
bution. This latter behavior was not surprising, as these
complexes were stabilized through charged residues, and
a few hydrophobic residues contributed to ligand coordi-
nation. Furthermore, these results suggested that the
more bulky the ligand was, the more favorable the LJ
interactions between the MCOs and the electron-donor
molecule were. Furthermore, the Tth-MCOm-ABTS
complex showed an opposite behavior, where the
electrostatic contributions guided the complex formation,
 Cavity-binding site of three bacterial multicopper oxidases 11
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Figure 5. Average distance between the electron-donor molecules and the histidines coordinating T1Cu. (A) Tth-MCO and its
complexes Tth-MCO-SGZ, Tth-MCO-3DM1 or Tth-MCO-3DM2. (B) CotA and its complexes CotA-ABTS, CotA-SGZ, and CotA-
3DM. (C) Cueo and its complexes Cueo-ABTS, Cueo-SGZ, and Cueo-3DM. (D) Cueom with its respective complex Cueom-ABTS.
(E) Tth-MCOm and its respective complex Tth-MCOm-ABTS.

suggesting that even this deleted form bound to the In contrast, an increment in the conformational mobility
ABTS. Notably, this idea must be examined through of the complex forms was observed for the Tth-MCO
co-crystallization studies for this system. Moreover, and Tth-MCOm complexes (Figure 6(A) and (D)) in the
although both CueO-ABTS and CueOm-ABTS show residues that form the ETBS (supplementary material
electrostatic interactions that stabilize their complexes, it (table 2S)), suggesting that the formation of these
is clear that there is almost twice the number of complexes could be entropically driven, as observed in
interactions for CueO-ABTS, suggesting better ligand other reports (Gutiérrez, Bello, Portillo-Téllez, Rodrí-
affinity for this complex. guez-Romero, & García-Hernández, 2013). However,
this behavior is opposite to that observed for the
Conformational mobility upon complex formation
Accumulated RMSF analyses for the equilibrated MD
simulations for the complexes studied here (see methods) Table 3. Interaction energies for different complexes.
show that the CotA, CueO, and CueOm complexes System Coul-SR (kJ) LJ-SR (kJ)
undergo a decrement in their conformational mobility
with respect to their unbound forms (Figure 6(B)–(D)). Tth-MCO-ABTS 49 ± 16 182 ± 21
A reduction in the conformational mobility was experi- Tth-MCOm-ABTS 331 ± 34 58 ± 18
Tth-MCO-SGZ 9±4 147 ± 10
enced not only for the residues forming the ETBS but Tth-MCO-3DM 3 ± 3first 60 ± 123DM1
also in the regions far from the ETBS (data not shown). 2 ± 2second 87 ± 133DM2
Furthermore, this behavior could contribute to the favor- CotA-ABTS 51 ± 9 170 ± 11
able enthalpy component through the favorable energetic CotA-SGZ 2±3 133 ± 16
CotA-3DM 1±2 85 ± 11
interactions and the reduction in the degrees of freedom
CueO-3DM 4±3 79 ± 12
upon complex formation, as observed in other MD simu- CueO-ABTS 800 ± 3 7 ± 28
lation studies (Bello, Gutiérrez, & Garcia-Hernandez, CueOm-ABTS 400 ± 73 21 ± 24
2012; Bello, Portillo-Tellez, & Garcia-Hernandez, 2011). CueO-SGZ 4±4 127 ± 14
 12 M. Bello et al.
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Figure 6. Accumulated RMSF for the free and bound forms of Tth-MCO, Tth-MCOm, CotA, Cueo, and Cueom. (A) Tth-MCO and
its complexes Tth-MCO-SGZ, Tth-MCO-3DM1 and Lth-MCO-3DM2. (B) CotA and its complexes CotA-ABTS, CotA-SGZ, and
CotA-3DM. (C) Cueo and its complexes Cueo-ABTS, Cueo-SGZ, and Cueo-3DM. (D) Tth-MCOm and Cueom with their respective
complex forms Tth-MCOm-ABTS and Cueom-ABTS, respectively.
Tth-MCO-ABTS complex, for which a reduction of the
conformational mobility was observed upon complex for-
mation (Bello et al., 2012). These results suggest that
complex stabilization is not entirely similar for the three
bacterial MCOs and that the ligand could be responsible
for the overall conformational mobility experienced upon
complex formation.
Conclusion
We characterized the successive binding modes of three
bacterial MCOs in native forms and truncated variants to
ABTS, SGZ, and 3DM. Although the electron-donor mol-
ecules initially explored different conformations during
simulation, the obtained complexes generated stable tra-
jectories that remained in the ETBS. An analysis of the
binding orientation, together with their energetic and
mobility contribution, demonstrated that for the majority
of complexes, the hydrophobic interactions were the most
abundant and played an important role in the determina-
tion of electron-donor molecule specificity. Furthermore,
a reduction in the conformational mobility of residues in
the ETBS and the extra binding site regions were
observed for most of the complexes, except for Tth-MCO
and Tth-MCOm complexes, which showed the opposite
behavior. For Tth-MCO-SGZ and Tth-MCOm-ABTS com-
plexes, both ligands were placed in a conformation that
might facilitate direct electron transference to H450
through D390 or D392, which are close to the ligands and
are followed in sequence by H393, as observed for the
Tth-MCO-ABTS complex (Bello et al., 2012; Enguita
et al., 2004). Furthermore, despite Tth-MCOm-ABTS
complex showing an opposite behavior with respect to
Tth-MCO-ABTS, where the electrostatic contributions
guided complex formation, the total energy was higher
for Tth-MCOm-ABTS, revealing that this mutant could be
a good candidate to perform crystallographic studies. For
the Tth-MCO-3DM complex, although two conformations
are present, only one conformation (3DM1), which was
located 7.4 Å from D392, could donate an electron to
D392, sequential in sequence to H393, which coordinates
T1Cu. Among the complexes generated using CotA, the
CotA-ABTS complex showed that its ligand was in a con-
formation that facilitated the direct transfer of an electron
to H497, which coordinates T1Cu. For the CotA-SGZ
complex, SGZ showed a conformation in which the donor
group was placed further from H497. However, there
were two charged residues (E496 and D499) localized
 Cavity-binding site of three bacterial multicopper oxidases 13

near SGZ at 6 and 8 Å, respectively, and both residues T1Cu Copper center type 1
were sequential in sequence with H497, which coordi- T2Cu Copper ion type 2
nates T1Cu. The results were similar for the CotA-3DM T3Cu Binuclear copper ion type 3
3DM 2,6-dimethoxy phenol
complex, in which 3DM is placed at a 9 Å distance from SGZ Syringaldazine
H497, and the only charged group involved in the elec- ABTS 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic
tron uptake was E496, located approximately 7 Å from acid
3DM. With respect to the complexes performed using RG Radius of gyration
CueO, the CueO-ABTS complex showed a ligand confor- SASA Solvent accessible surface areas
LJ Lennard-Jones
mation in which electron transfer to T1Cu could only be Coul Coulomb energy
achieved through the cluster of methionines involved in
putative Cu+1/Cu+2 traffic. For the CueO-SGZ and CueO-
3DM complexes, both the ligand conformations placed Acknowledgments
the donor group at approximately 12.5 Å from H505.
This work was supported in part by DGAPA, UNAM [PAPIIT,
However, a charged residue (E476) near the donor group
IN 204611, CONACyT [Grants 102370, 128156 and
of either electron-donor molecule could participate in the 132353] and ICyTDF (PIRIVE09-9, COFAA-SIP/IPN). MB
uptake and subsequent transfer of an electron to H505. acknowledges the Program of Postdoctoral Scholarships from
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The CueOm-ABTS complex showed a ligand conforma- DGAPA (UNAM) and CONACyT. This work was carried out
tion in which the donor group was approximately 16.5 Å using a KanBalam supercomputer, provided by DGTIC, UNAM.
from H505, and despite the truncation of α-helix 4, a
group of methionines remained that, in coordination with Supplementary material
E476, might participate in the electron transfer to H505. The supplementary material for this paper is available
In addition, although both CueO-ABTS and CueOm- online at http://dx.doi.10.1080/07391102.2013.817954.
ABTS were stabilized through electrostatic interactions,
the total energy stabilizing the CueO-ABTS complex was
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