Insights in 17b-HSD1 Enzyme Kinetics and Ligand
Binding by Dynamic Motion Investigation
Matthias Negri1,2, Maurizio Recanatini3, Rolf W. Hartmann1,2*
1Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbru ¨cken, Germany, 2Helmholtz Institute for Pharmaceutical Research Saarland, Saarbru ¨cken,
Germany, 3Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
Background: Bisubstrate enzymes, such as 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1), exist in solution as an
ensemble of conformations. 17b-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially
attractive target for breast cancer treatment.
Methodology/Principal Findings: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all
available crystal structures was performed and representative conformations were assigned to each step of the putative
kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed
using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17b-HSD1 as
starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone,
respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more
accurately which of the MD complexes describes a specific kinetic step.
Conclusions/Significance: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-
complex approach revealed an essential role played by the backbone and side chain motions, especially of the bFaG9-loop,
in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17b-HSD1, where ligand-binding induced
concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway.
Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17b-HSD1 and
we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17b-HSD1,
as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also
characterized by a rigid core and a flexible region directing their catalysis.
Citation: Negri M, Recanatini M, Hartmann RW (2010) Insights in 17b-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation. PLoS ONE 5(8):
Editor: Floyd Romesberg, The Scripps Research Institute, United States of America
Received March 29, 2010; Accepted July 6, 2010; Published August 10, 2010
Copyright: ? 2010 Negri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Cytoplasmic proteins are present in solution as an ensemble of
conformations, which are in a dynamic equilibrium, strongly
influenced by the presence of ligands (in case of enzymes:
cofactors, substrates, inhibitors, or other proteins). This is
especially true for enzymes following a bisubstrate kinetic, like
Escherichia coli dihydrofolate reductase (DHFR)  and human 17b-
hydroxysteroid dehydrogenase type 1 (17b-HSD1 or SDR28C1,
according to the new nomenclature ; E.C. 18.104.22.168), where
concerted dynamic motions are necessary between the enzyme
conformations responsible for specific kinetic steps. An in-depth
knowledge of both protein dynamic and its influence on ligand
binding could effectively speed up rational drug design . These
new drugs might act not only by competing with the substrate for
its binding site, but also by inducing a dynamic dysfunction of the
enzyme by hindering the switch between its conformations .
In estrogen target cells 17b-HSD1 catalyzes the NADPH-
dependent reduction of estrone (E1) to the biologically highly
potent 17b-estradiol (E2) [4–6] (Figure 1). It has been shown that
in post-menopausal women with hormone-dependent breast
cancer tumor proliferation is driven by increased levels of E2
[7–8]. As 17b-HSD1 is often overexpressed in breast tumor cells, it
is considered as a novel therapeutic target [9–12].
Recently, Cooper et al. elucidated the complete kinetic
mechanism for the rat liver 3a-HSD and could assign different
enzyme forms to the specific reaction coordinates . 3a-HSD
and nearly all other HSD enzymes are described to follow a
sequential ordered bi-bi kinetic mechanism, where the cofactor
enters first and exits last. The kinetic mechanism of 17b-HSD1 is
still not fully clarified, although it has been reported to follow a
rapid equilibrium random bi-bi mechanism (Figure S1), a
peculiarity compared to other HSDs [14–16]. The high
NADPH/NADP+gradient (.500:1) and the excess of NADPH
with respect to E1 in vivo, as well as the thermodynamically
favoured NADPH oxidation [17–18], suggest the presence of
NADPH in the enzyme prior to steroid binding. Asn114, Ser142,
Tyr155 and Lys159 form the catalytic tetrad of 17b-HSD1 ,
conserved in many HSD enzymes, and are involved in the pro-S
hydride transfer from NADPH to the a-face of the C17 carbon as
well as in the proton transfer between the OH group of Tyr155
and the C17 oxygen of E1  (Figure 2).
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Remarkably, the catalytic reaction of 17b-HSD1 is reversible in
vitro; but it is unidirectional in vivo, because of the abundance of
NADPH. No crystal structure of the ternary complex enzyme (E)-
NADPH-E1 exists. We think that possible reasons for that might
be problems in the crystallization process, i.e. low electron density
for E1, but also the very fast transfer rate observed for the
reduction of E1 (kcat1.5 s21for dimer ; 2.9 s21). E2 has a
two order of magnitude reduced binding affinity to 17b-HSD1
(KM4.6 mM) compared to that of E1 (KM0.03 mM), but a similar
(less reduced) kcatof 2.0 s21. However, discordant kcatand
KMvalues are reported in literature for both E1 and E2, in part
depending on the cofactor used for the experiment (NADPH or
NADH), its concentration (20 or 50 mM), etc. [14–15,18,20–22]
The kcat and KM values reported here were obtained using
NADP(H). Due to the excess of NADPH over NADP+and due to
the marked specificity of 17b-HSD1 for the E1 reduction using
NADPH instead of NADP+as cofactor (about 240-fold higher
than for E2 oxidation) , we prospect that in vivo the backward
reaction E2 to E1 does not take place. Moreover, the KMof E1 is
12-fold lower when using NADPH (0.03 mM) than when using
NADH (0.36 mM), hence underlining the crucial role played by
the third phosphate group of the cofactor in the E1 reduction .
Figure 1. Reduction from E1 to E2 and non-steroidal and steroidal inhibitors. The substrate E1 is colored in blue, the cofactor NADPH in
red. Amino acids, which are either involved in the catalysis or are responsible for ligand/cofactor stabilization, are colored in black and residue
labelled. The hydrogen bonds are in dashed lines while the proton transfers are highlighted with arrows.
Figure 2. Postulated catalytic mechanism of 17b-HSD1. The substrate E1 is represented in blue, the cofactor NADPH in red. The amino acids,
which are either involved in the catalysis or are responsible for ligand/cofactor stabilization, are colored in black and residue labelled. The hydrogen
bonds are in dashed lines while the proton transfers are highlighted with arrows.
Kinetic Cycle of 17b-HSD1
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Steroidal and non-steroidal inhibitors of 17b-HSD1 have been
reported (Figure 1) [9–11,23–29]. The former mimic the substrate
and are supposed to bind in the substrate binding site (SUB), a
narrow hydrophobic tunnel . They are stabilized by hydrogen
bonds with Tyr155/Ser142 and His221/Glu282, located on both
ends of the pocket, and, further, by hydrophobic contacts 
Recently, Poirier et al. published a series of hybrid inhibitors,
based on a E1/E2 core with substituents of various lengths in C16
position . These inhibitors occupy both the SUB and the COF
(cofactor binding site), competing with E1 and NADPH, as
demonstrated by the binary complex E-HYC (PDB entry 1i5r). Up
to now no crystallographic data exists describing the binding mode
of non-steroidal inhibitors, although
performed by various groups suggested some classes of non-
steroidals to bind like their steroidal analogues in the SUB [9–
11,27–29] and others to occupy only partially the SUB protruding
into the COF, like compound 1 .
Herein, we present the first, to our knowledge, computational
investigation of the kinetic mechanism of 17b-HSD1, one of the
thirteen 17b-HSDs belonging to the short-chain dehydrogenases/
reductases (SDR), a large superfamily of NAD(P)H-dependent
enzymes. Furthermore, the linkage between representative enzyme
conformations and the five steps described for the random bi-bi
mechanism, an in-depth computational analysis of the flexible
bFaG9-loop, and its role in enzyme catalysis and in ligand binding
were elucidated. Thus, a multi-trajectory/-complex molecular
dynamic (MD) strategy was followed, based on four different
enzyme conformations. Molecular mechanics Poisson-Boltzmann
surface area (MM-PBSA) methods , in combination with
normal-mode analysis (NMODE) , were exploited to calculate
the free energies of the complexes with the aim to substantiate our
kinetic hypothesis and to determine the best enzyme conformation
for binding of E1 and NADPH, respectively.
Moreover, we analyzed the kinetic mechanism of 17b-HSD1 by
means of MD simulations in order to enlarge the conformational
sampling of the enzyme structures. This ensemble of conformers
should be used to improve drug-design through the identification
of new inhibitor binding modes. In particular, making a parallel to
E. coli DHFR which also has a bi-bi kinetics, our long range goal is
to design 17b-HSD1 inhibitors able to ‘‘freeze’’ the enzyme in one
of its kinetic substates, hindering the evolution of the kinetic cycle,
in a similar way as methotrexate (MTX) does in DHFR .
Results and Discussion
1. Structural analysis of human 17b-HSD1 crystal
17b-HSD1 is a homodimeric protein and shows the typical rigid
b-a-b fold of the SDR-family with a core of parallel b-strands
fanning across the center and a-helices draped on the outside
(Figure S2) , resulting in a rigid COF at the N-terminus and a
structurally variable C-terminus, hosting the SUB . The
structurally conserved region comprises Rossman fold and
GxxxGxG motif (G is glycine and x any other residue),
characteristic of oxidation/reduction enzymes that bind nicotin-
amide cofactors, plus the YxxxK sequence (Y is Tyr155 and K
Lys159) that participates in catalysis . In sharp contrast to
other 17b-HSDs, type 1 is characterized by a long, flexible bFaG9-
loop and a C-terminal helix, both delimiting the SUB and
probably involved in enzymatic catalysis and in entrance and exit
mechanisms of small molecules.
Until May 2010 twenty crystal structures of 17b-HSD1 were
available in the Protein Data Bank (PDB) as apoform, binary or
ternary complex (Table 1). The loop residues Thr190-Gly198 of
the PBD entry 1fdt  were modelled in two different
conformations and had been considered as two different
conformers, denoted hereafter as 1fdtA and 1fdtB. Superimposi-
tion of all crystals gave a mean backbone RMSD (root mean-
square distance) of ca. 0.5 A˚(all-atom RMSD ca. 1.5 A˚). The only
flexible part was identified in the above mentioned bFaG9-loop,
which could adopt different conformations, strongly modulating
shape and volume of the active site (including both COF and
SUB). Eight of these crystal structures were incomplete, missing
mainly the loop residues Phe192-Val196, while the remaining
eleven showed high b-factor values for this area, an additional hint
for its flexibility. In the fully resolved structures the loop occurred
as a disordered random coil. Moreover, the loop was found in a
stable orientation only for ternary complexes, which had enzyme,
cofactor and steroidal product or inhibitor fully resolved (1a27,
1fdtA, 1fdtB, 1equA, 1fduC and 3hb5; cluster cl2 in Figure 3). For
all other crystal structures no univocal loop conformation could be
ascribed to the presence of either cofactor or ligand. Nevertheless,
this multiplicity of conformations suggested various substates for
17b-HSD1, which are subjected to fast dynamic motions and
secondary structure rearrangements of the loop. Most of the
binary complexes E-E2 were fully resolved, while the E-NAD(P)+
complexes lack part of the bFaG9-loop and no binary complex
with NADPH or with E1 existed at all (Table 1). The existence of
numerous fully resolved E-E2 complexes led us to speculate that
NADPH became tightly bound to the enzyme only after the
steroid had entered the cavity and the loop had changed its
conformation. Interestingly, a similar behaviour was confirmed
also by molecular dynamics of some complexes, as it will be
elucidated later in this article.
The twelve full-length crystal structures of 17b-HSD1 were
superimposed and clustered according to the backbone RMSD
(mean value ca. 3 A˚) of the five loop residues Phe192-Val196.
Three clusters cl1, cl2 and cl3 (Figure 3; representative structures:
1iol, 1a27 and 1i5r, respectively) could be identified, as well as the
presence of three gates, two close to the loop (gate 1 and 2) and a
third one (gate 3) close to the C-terminal helix (Figure 4).
However, this backbone classification disregarded active site
volume changes and was limited to ascribe the presence of opened
gates for the three clusters cl1–cl3. Thus, clustering was repeated
taking into account all atoms of the same loop residues, which
resulted in a mean RMSD value of ca. 6 A˚. Five clusters could be
identified (Figure 4 and S3) and Phe192, in particular, emerged as
an important marker for the conformational variations, since its
side chain presented the largest RMSD deviation (ca. 10 A˚)
rotating for about 200 degrees around the loop axis (Figure S4 and
Video S1): it turned from the inner cavity, where it occluded the
SUB and stabilized the substrate in the correct position for hydride
transfer (1fdtB), toward the outside, where it chaperones E1/E2 or
the cofactor in and out of the active site (1fdtA). These two
extremes in the orientation of the side chain of Phe192
corresponded to the major changes of the active site volumes of
all wild type E-NADPH-E2 complexes, where the volume ratio
SUB/COF changed from 30:70 in 1fdtB to 37:63 in 1fdtA
2. Which mechanisms govern the kinetic cycle of
17b-HSD1 is described to follow a random bi-bi kinetic
mechanism (Figure 4, S1 and S3) [14,16]. Based on biochemical
and structural data (i.e. the excess of NADPH over E1 and the
different crystal structures for the apo-, holo- and ternary form) we
hypothesized 17b-HSD1 to follow a five-step preferential pathway
in vivo. Moreover, we assigned enzyme conformations to the
Kinetic Cycle of 17b-HSD1
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specific steps and tried to rationalize the reaction pathway
according to the presence of cofactor, E1 or E2, as well as to
the conformational changes expected to occur in order to allow the
kinetic cycle to evolve further (in brackets the PDB id of the crystal
structures best describing the starting conformer for each specific
2.1. Step 1 (1bhs).
We postulate that NADPH will bind first
and, in particular, into the COF, when the bFaG9-loop is placed
in cluster cl2/1, with gate 1 wide and accessible. The entrance of
the cofactor and its interactions with the apoform of the enzyme
might induce a conformational rearrangement of the flexible loop
toward the COF, with the loop moving from cl2 into cl3, thus
closing gate 1 and opening gate 2. This loop-motion is intended to
favour the entrance and binding of E1.
2.2. Step 2 (1i5r).
E1 enters and the loop slides back to its
starting point in cluster cl2/1, gate 2 will be closed again and the
next step reached.
2.3. Step 3 (1fdtB, 1a27).
After the bFaG9-loop has allowed
the entrance of E1, it orients Phe192 and Met193 inward in order
to stabilize the substrate in an optimal position for the pro-S
hydride transfer. The first ternary complex (E-NADPH-E1) is
formed and the catalytic reaction takes place.
Table 1. Summary of the 20 (21) crystal structures of 17b-HSD (April 2010).
backbone cluster of
active site volume
(A˚3) calculated with castP
(loop helix)COF SUB
1a27  285 E2, NAP1.90A cl2– 1855
1bhs  284–2.20A cl1– 2214
1dht  284 DHT 2.24A cl1– 1944
1equ  284EQI, NAP3.00A/B cl2 (A)/cl1 (B)– 2403
1fds  282 E21.70A–– 2323
1fdtA  285E2, NAP2.20A cl2– 2000
1fdtB  285 E2, NAP2.20A cl2– 2259
1fdu  281 E2, NAP 2.70 A–Dcl2 (C)H221LC: 1645
1fdv 285 NAD3.10A–D cl2 (A, C)H221L A: 1892/C: 2051
1fdw  279 E22.70A– H221Q2301
1i5r  285 HYC1.60A cl3– 2755
1iol  284E2 2.30A cl1– 2296
1jtv  278TES1.54A–– 1855
1qyv  276NAP1.81A–– 2906
1qyw  2765SD, AP21.63A–– 1858
1qyx  277 ASD, AP21.89A–– 1888
3dhe  284 AND2.30A cl1–2152
3dey  277 DHT1.70A––1882
3hb4  284E2B2.0X cl2– 2707
3hb5  284 E2B, NAP2.0Xcl2– 2179
3klm  277DHT 1.70A–– 1882
acomplexed ligands: E2 (estradiol), NAP (nicotinamide-adenine-dinucleotide phosphate; NADP+), DHT (dihydrotestosterone), EQI (equilin), NAD (nicotinamide-adenine-
dinucleotide), HYC (O59-[9-(3,17b-Dihydroxy-1,3,5(10)-estratrien-16b-yl)-nonanoyl] adenosine), TES (testosterone), 5SD (5a-androstane-3,17-dione), ASD (4-androstene-
3,17-dione), AP2 (29-monophosoadenosine 59-diphosphoribose), AND (3b-Hydroxy-5-androstene-17-one).
bbFaG9-loop backbone clusters: (cl1) loop close to the aG9-helix, (cl2) loop placed in front of the nicotinamide moiety of the cofactor, (cl3) loop shifted towards the
(Resid) total number of amino acids crystallized; (Res) resolution of the crystal structures in A˚.
Kinetic Cycle of 17b-HSD1
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2.4. Step 4 (1fdtA, 1iol).
drift into cluster cl1, protruding more into the SUB and opening
gate 1. This gate rules the egress path for NADP+, hence when it is
open the oxidized cofactor can leave the active site. The steroid,
however, is still stabilized in the SUB, shielded by Phe192 and
2.5. Step 5 (no crystal structure given).
leaves the active site through gate 3.
Interestingly, for all five substates the loop residues and Phe226
on the aG9-helix are oriented in a way to avoid solvent
accessibility to the SUB.
Now the flexible loop is expected to
Herein the steroid
3. Validation of the catalytic cycle by means of molecular
MD simulations have been proven to be very useful tools in the
analysis of dynamic motions of enzymes with flexible loops, which
might play a prominent role in either the catalytic cycle and/or
ligand binding. In particular, kinetic cycle and ligand-binding
related motions of E. coli [1,3] have been intensively studied by
means of both experimental and computational methods. While
the former (such as site-directed mutagenesis, isothermal calorim-
etry, NMR dispersion, crystallography, etc.) constituted the basis
for the understanding of DHFR functionality and catalytic
mechanism, the computational methods (such as MD simulations,
thermal fluctuation, free energy calculations, etc.) were applied
with success in order to elucidate protein plasticity and allosteric
mechanism. Moreover, they were envisaged to address new
strategies in drug design, as for example the inhibition of E. coli
DHFR by functional disruption, where the enzyme is frozen in a
conformer that stops the kinetic cycle .
Herein, we applied MD simulations and free energy calculations
pursuing as a main goal to substantiate the assignment of different
enzyme conformations of 17b-HSD1 to the five steps encountered
along its catalytic pathway. Moreover, we wanted to investigate
transition steps and enzyme motions influenced by starting
conformation and ligand binding. To this end, eight MD
simulations (models I-III; Text S1) were designed based on the
crystal structures 1fdtA, 1i5r, 1fdtB, and 1bhs, which differ
strongly in the orientation of the bFaG9-loop (Figure 3, 4 and S3).
In fact, the enzyme was investigated in its opened-state (1fdtA) -
characterized by a fused COF and SUB and by Phe192 turned
outward, in a semi-opened/occluded state (1i5r) - with the loop
shifted toward the COF and Phe192 more buried than in 1fdtA,
and in its closed-state (1fdtB) - mimicking the catalytic moment
with a closed SUB and Phe192 pointing inward perpendicular to
the catalytic Tyr155. The MD simulations of apoform E (A),
binary E-NADPH (B) and ternary complexes E-NADPH-E1 (C) of
17b-HSD1 were evaluated in terms of geometry (RMSD) and
energy stability of the complexes, ligand and secondary structure
displacement with respect to the starting pose, and the time
required to reach a stable RMSD and energy plateau. For each
stable sector of the MD trajectories absolute free energy (DG) and
relative binding affinity (DGbind) were calculated by means of MM-
PBSA methods and NMODE analysis. Thus, in order to
determine whether a preferential enzyme conformation exists for
the binding of NADPH and E1, we compared DG and DGbind
values of the binary complexes B1–B3 and of the ternary C1–C3,
respectively (Table 2).
3.1 Step 1.
The MDs performed for step 1 pursued two main
goals. First, we wanted to identify the enzyme conformation best
representing the enzyme in its apoform and, thus, the folding that
the flexible bFaG9-loop will adopt in absence of other ligands. And
second, we expected to find an easily accessible COF, and
eventually SUB, in the stable parts of the trajectories, as a
consequence of opened gates 1 and 2.
Two MD simulations were performed with the enzyme only
(1bhs – A1, 1fdtB – A2; Figure S5). In the apoform crystal
structure 1bhs (A1) the fully resolved bFaG9-loop laid in cluster
cl1, presenting a turn-helix-turn motif and the side chains of its
residues oriented along its axis. Thus, a 12 ns MD A1 was
performed in order to verify the reliability of 1bhs as step 1
enzyme conformation. Remarkably, the loop evolved toward
cluster cl2, shielding the COF and especially the area where the
nicotinamide moiety is found. This led to a large active site
suitable to host both cofactor and ligand. The enzyme showed the
tendency to preserve the hydrophobic character of the SUB,
turning the side chains of the apolar residues (mostly Phe192,
Phe226 and Phe259) inward, which occluded the SUB and
avoided water to be placed in there. Met193 was also deeply
buried in the SUB, occupying the space where steroids are
normally placed. On the contrary, all polar residues belonging to
the FG segment and to the C-terminal helix turned outside
forming ion-pairs responsible for the closure of gate 2 and 3. The
final stable complex of this MD was considered as a transition state
of step 1, with an accessible COF, suitable to host NADPH, and
an occluded SUB, not ready to accommodate the substrate.
According to our hypothetic cycle, the bFaG9-loop should drift
now further toward the COF (i.e. into cluster cl3), in concert and/
or slightly delayed to NADPH entrance, resulting in a progressive
closure of the COF (closed gate 1) and accessibility of the SUB
(opening of gate2; 1i5r).
The second MD A2 was performed on the apoform of the
closed-state conformation 1fdtB, stripped of both cofactor and
steroid. The aim was to investigate whether the starting enzyme
conformations of A1 and A2 would lead to different final tertiary
structures or if they could converge to a common folding and loop
axis orientation. However, already after 1 ns of simulation a
marked change in the tertiary structure of the 1fdtB apoform was
Figure 3. Clusters according to backbone RMSD. Representative
structures of the three bFaG9-loop conformations cl1 (yellow, 1iol), cl2
(magenta, 1a27) and cl3 (blue, 1i5r), clustered according to the
backbone RMSD of the five residues Phe192-Val196. bFaG9-loop, aG9-
helix (green) and C-terminal helix (cyan) are rendered as cartoon,
NADPH and E1 as sticks and the rest of the enzyme (active site) as
Kinetic Cycle of 17b-HSD1
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observed involving FG segment and C-terminal helix. Phe192
remained turned inward, while Met193 rotated out toward the
aG9-helix, becoming responsible for the disruption of the
The two simulations did not converge to a common
conformation; moreover, the final complex of A2 resulted in a
partially unfolded structure. This result suggested that the presence
of cofactor and/or ligand would be necessary to assure a correct
Table 2. Free energy calculations for the MD simulations B1–B3 and C1–C3.
E PDB MD codeELECVDWGAS PBSOLPBTOT (DGbind) TSTOT
meanmean mean mean mean (±SE) mean (±SE) mean
Model II - E-NADPH
Model III - E-NADPH-E1
DG and DGbindvalues correspond to the longest stable RMSD plateau for each MD; i.e. the values for C1 and C3 correspond to the plateaux until ca. 10 ns, while the
free energy values for the extended (+6/7 ns) section of the MDs are reported in the text.
(E) starting conformation of the enzyme; (DG) absolute free binding energy; (DGbind) (PBTOT) relative free binding energy; (ELEC) electrostatic contribution in gas
phase; (VDW) Van der Waals contribution in gas phase; (GAS) free energy in vacuum; (PBSOL) solvation energy; (TSTOT) (TDS) entropic contribution; (mean) mean
value; (SE) standard error of the mean; all energies expressed in kcal/mol.
Figure 4. Random bi-bi kinetic cycle of 17b-HSD1. To each step one or more crystal structures were assigned. Side chains of the bFaG9-loop
residues Phe192-Lys195 are rendered as sticks and the orientation of the loop (blue ball) with respect to the aG9 helix (in gray) is shown as cartoon.
The arrows 1–3 indicate the entrance and egress gates identified along the reaction pathway. 1fdtA is not included in the balloon of step 4 due to its
different loop axis orientation with respect to 1iol.
Kinetic Cycle of 17b-HSD1
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transition between the five steps of the enzymatic cycle and that
the choice of the starting conformation might be crucial as well.
The stable trajectory of A1, however, represents a nice simulation
of step 1, especially if the exposed COF is considered.
3.2. Step 2.
In step 2 the COF should already be occluded
and gate 1 being closed by the motion of the flexible loop into cl3.
The loop should stabilize NADPH in the COF, and also maintain
the substrate in the SUB in a correct position for the hydride
transfer. The latter requires the loop to float again into cluster cl2,
thus centrally placed.
Three 9 ns MDs were carried out on the binary complex E-
NADPH (B) and another three, lasting 10 ns, on the ternary
complex E-NADPH-E1 (C), with the enzyme in its opened (1fdtA -
B1, C1), semi-opened (1i5r - B2, C2) and closed (1fdtB - B3, C3)
state, respectively. The aim was to obtain insights into the role
played by the loop in the stabilization of NADPH, the shielding of
the SUB and the stabilization of E1.
Simulation B2 (1i5r-NADPH) was characterized by a quickly
reached, very long stable RMSD plateau (Figure S6), indicating a
good stability of the complex. Also the loop showed a stable
RMSD profile, although, while the lower part (Glu194-Ser199)
still remained close to the cofactor in cluster cl3, the upper part
(Thr190-Met193) drifted toward the aG9-helix. This seemed to
occur as a direct consequence of the outward rotation of Phe226,
which was responsible to sustain the nicotinamide moiety in the
starting complex, but which was also involved in opening gate 2, a
prerequisite for E1 entrance. The loop was folded to a short helix
and stabilized by the ion-pair Lys195 and 29-phosphate of the
cofactor, which trapped the adenosine moiety. In the final
structure the upper part of the loop, placed in cluster cl2,
occluded part of gate 2, reducing the solvent accessible surface and
preserving the hydrophobic character of the SUB. Nevertheless, a
hydrophobic tunnel remained open between loop and helix,
suitable for steroid transition. The complex stability of B2 was also
reflected by a good free energy DG (29.35 kcal/mol) with respect
to the positive values of B1 and B3 (Table 2), which substantiated
our structural hypothesis for catalytic step 2.
The MD of the ternary complex 1i5r-NADPH-E1 (C2)
described very well the expected loop motion from cluster cl3,
shielding the COF, to cluster cl2 (Figure 5). Despite a suboptimal
DG (20.96 kcal/mol; but with a good DGbindof 223.68 kcal/
mol)compared tothat of the
(28.31 kcal/mol), we considered the MD C2 as a valuable
simulation of step 2 from a binary state to the ternary complex
(Figure 5B). Effectively, this simulation ended with a stable
complex in which E1 was properly bound into the SUB, involved
into an h-bond network with Ser142/Tyr155 and His221/
Glu282. Furthermore, the bFaG9-loop drifted into cl2 shielding
the active site and forming a short a-helix, characteristic for most
of the ternary complexes present in the PDB.
3.3. Step 3.
In this step the hydride transfer is meant to take
place, thus no major tertiary structure rearrangements were
The three MDs of the ternary complex E-NADPH-E1 (C1–C3;
Figure S7) highlighted very well the important role of Phe192 and
Met193 in stabilizing E1 close to the cofactor to ensure an optimal
distance necessary for hydride transfer. When these two residues
were turned outward, as it happened for the opened and the semi-
opened complexes 1fdtA-NADPH-E1 (C1) and 1i5r-NADPH-E1
(C2) respectively, E1 had more space to move at the beginning of
the simulations and resulted less stably bound.
The catalytic residues Ser142, Tyr155, and Lys159 showed
small RMSD values for all three ternary complexes, even after
10 ns of dynamics (Figure S7D). This observation was in
accordance to experimental site-directed mutagenesis studies
, where the enzymatic activity was abolished because of the
mutation of one or more of these residues. Moreover, the
conformational stability of these residues suggested them to be
essential for the postulated catalytic mechanism (Figure 2) ,
while other regions, for example the bFaG9-loop, to be the driving
force that keeps the catalytic cycle running. The trajectories
observed for the FG-segment residues Phe192 (in particular),
Lys195 and Phe226 in the three simulations C1–C3 overlapped
nicely with their orientations in the eighteen enzyme conforma-
Figure 5. Motion of the bFaG9-loop in MD C2 (1i5r). (A) The overlay of multiple snapshots of the trajectory C2 showed the transition of the loop
axis from cluster cl3 to cl2 (color-coded in a range from red to blue, according to start and end of the MD, respectively). (B) Important active site
residues of the initial (violet, step 1), stable (orange, step 2) and final complex (yellow) are rendered as sticks.
Kinetic Cycle of 17b-HSD1
PLoS ONE | www.plosone.org7 August 2010 | Volume 5 | Issue 8 | e12026
tions (Figure S4) and fitted very well with the conclusion of Mazza
et al. , who suggested a direct involvement of Lys195 and
Phe192 in the catalytic cycle.
Phe192 was meant to play a very important role not only in
steroid stabilization, but also in the catalytic reaction itself. In most
ternary complexes with E1, Phe192 pointed perpendicular toward
Tyr155. In this T-shaped conformation, Phe192 is likely to
increase the acidity of the phenol group of the catalytic Tyr155
from a pKaof ca 9 to 6 , which presumably might facilitate the
hydride transfer to the estrone. The final pose of Phe192 in C2
and C3 was consistent with this observation and enforced its role
in protecting the nicotinamide moiety and activating the catalysis.
3.4. Postulated entrance mechanism of E1.
structures of the opened and the closed complexes C1 and C3
(1fdtB-NADPH-E1), respectively, presented the loop axis (main
chain) in similar starting conformation (cluster cl2), but with the
side chains of the loop residues oriented opposite. After ca. 9 ns
the MD C1 presented a long, stable RMSD plateau (Figure S7B).
However, when this simulation was prolonged to ca. 16.5 ns, the
all-atom RMSD rose to 3.5 A˚, mostly related to bFaG9-loop
motions (Figure 6). In fact, the extended part of C1 was
characterized by slow concerted motions of the FG-segment and
the C-terminal helix, i.e. increasing loop RMSD in Figure 6B,
which led to the opening of gate 2 (the loop moves toward the
COF into cl3) and gate 3. Remarkably, concomitant to these
rearrangements the DG increased from 22.6 (after 9 ns) to
26.02 kcal/mol (after 16.5 ns). A progressive loss of the hydrogen
bond between E1 and Ser142 was observed in concert with the
outward rotation of Met193, Phe192, and Phe226. Thus, E1
drifted beneath the cofactor, turning its D-ring toward gate 2. A
‘‘backward analysis’’ of the whole simulation C1, ending with a
conformation similar to the starting one of C3 (1fdtB), led us to
postulate a ‘‘pull-and-push’’ mechanism for E1 entrance (Figure
S8): E1 enters the binding site through gate 2, pulled by the inward
rotation of Lys195 to which the steroid is bound with the C17
carbonyl. This bond will be released once E1 is placed into the
SUB and Lys195 could rotate further toward the 29-phosphate of
the cofactor to stabilize it. Thus, Phe192 turns inward drifting
below the nicotinamide ring of NADPH pushing hereafter E1 into
the SUB and reaching so a similar conformation as the starting
structure of C3 (1fdtB-NADPH-E1). Other residues delimiting
gate 2 might also adapt to the steroid, as for example Phe226,
which turns from the outside to the inner cavity stabilizing E1
(stable complex of MD C3; in blue in Figure 7).
3.5. Step 4.
In step 4 we expected the bFaG9-loop to drift
further toward the aG9-helix into cluster cl1, protruding slightly
into the SUB and opening gate 1, and, as a direct consequence,
that NADP(H) leaves the COF via gate 1.
Two out of the six binary and ternary MD simulations were
meaningful for describing this step: 1fdtB-NADPH-E1 (C3), which
confirmed the loop motion with subsequent opening of gate 1 and
the drift of E2 toward gate 3; and 1fdtA-NADPH (B1), which
showed the egress path of the cofactor due to gate 1 enlargement.
Although at this stage the steroid should be E2, we considered
for step 4 the simulations C1–C3, where E1 and NADPH were
used instead of E2 and NADP+. We consider this approximation
as acceptable, because no differences between the hydrogen bond
patterns of E1 and E2 with 17b-HSD1 exist and because forcefield
based methods, such as MD simulation, are not sensitive enough
to handle the electrostatic differences between ligand and cofactor
forms . Moreover, in this study we focused on the role of
bFaG9-loop residues in substrate binding and on the determina-
tion of protein-ligand binding thermodynamics, and not on the
simulation of the hydride transfer in the E1-E2 reduction. For the
latter high-level quantum chemical calculations would be required,
capable of dealing with bond-formation and –breaking as well as
of treating electrons explicitly, in contrast to force field-based
methods which handle atoms. In all complexes, E1 was placed in
the same pose, analogous to that of E2 in the crystals. As already
seen, all the MDs C1–C3 reached a stable RMSD plateau after
already 4 ns (Figure S7), maintaining E1 placed in the SUB, but
the DG values for their stable segments varied notably (Table 2).
The MD of the ternary complex C3 best described step 4 with an
DG of 28.31 kcal/mol for the first 10 ns (DGbind228.3 kcal/
mol): E1 maintained its hydrogen bonds to Ser142/Tyr155 for the
first 10 ns, until being pushed toward gate 3 and out of the SUB in
the following 7 ns due to the loop motion toward the aG9-helix
(Figure 7) and the inward rotation of Phe192 and Phe226.
On the other hand, in simulation 1fdtA-NADPH (B1), character-
ized at its beginning by a wide access to the COF due to the outward
Figure 6. RMSD analysis of the last 7 ns of MDs C1 and C3. (A) Time-dependent all-atom RMSD for all residues of the ternary complexes C1
(1fdtA) (light green) and C3 (1fdtB) (red) and (B) for the bFaG9-loop residues 187–199 of C1 (dark green) and C3 (orange).
Kinetic Cycle of 17b-HSD1
PLoS ONE | www.plosone.org8 August 2010 | Volume 5 | Issue 8 | e12026
rotated Phe192, we observed a concerted opening of gate 1 and
closing of gate 2 due to the shift of the loop axis toward the aG9-helix.
Thus, after a gradual increase in the first 5 ns, the all-atom RMSD
reached a plateau and maintained it until the end of the simulation.
Differently, the cofactor RMSD continued to rise (Figure S6D), in
accordance to the movement of NADPH out of the COF, which was
facilitated by the outward rotated Arg37, not burying the adenosine
moiety in the COF anymore. Notably, the cofactor-stabilizing role of
Arg37 had been previously demonstrated by site-directed mutagen-
esis studies . The aG9-helix rotated slightly rightward for about
30 degrees, adapting its residues to those of the loop. In this motion,
His221 moved outward, breaking the salt bridge with Glu282 and
enlarging gate 3. The free energy of complex B1 (+35.53 kcal/mol)
clearly indicates that this enzyme conformation is disfavoured to
stabilize NADPH, which ends up solvent exposed, suggesting
complex B1 to be a good model to simulate the exit of the cofactor
in step 4.
3.6. Step 5.
At this stage we expected E2 (in our case E1) to
exit the SUB via gate 3 as well as a concerted rearrangement of the
FG-segment residues to shield the empty SUB.
MD simulation C3 was prolonged up to ca 17.5 ns, with the
purpose to evaluate the propensity of E1 to escape the active site.
While the all-atom RMSD of the extended part of C3 (10–17.5 ns)
decreased (Figure 6A), its loop RMSD increased slowly (Figure 6B).
This sector of the dynamic was characterized by the closure of gate
2, the opening of gate 3 and the concerted breaking of the
hydrogen-bond network between E1, His221 and Glu282. In fact,
in C3 E1 drifted toward gate 3 and stuck out of the SUB. The
progression of E1 to the end of the tunnel was favoured by the
loop motions, the rotation of the apolar residues Phe192, Met193
and Phe226 into the SUB (Figure 7), and by the edging out of
His221 and Glu282 and the consequent enlargement of gate 3.
Interestingly, a decrease of DG from 28.31 (after ca. 10 ns) to
26.36 kcal/mol (after 17 ns) was observed.
Ideally, the final structure of step 5 should be similar to the
starting one of step 1. This could not be reached with our 10 ns
MDs. The reasons therefore could be the presence of E1 in the
SUB, influencing the loop motions, but also because a much
longer simulation time would be required to sample such marked
conformational changes. However, the final structure of C3
reveals opened gates 1 and 3 and an occluded gate 2, with the
bFaG9-loop close to the aG9-helix, as observed for 1bhs, the
representative crystal for step 1.
Very recently, a binary and a ternary complex of 17b-HSD1
(PDB entry 3hb4 and 3hb5) with the potent E2-derived inhibitor
E2B were published . In the ternary structure the bFaG9-loop
was oriented in cluster cl2, in analogy to all other ternary
complexes, confirming its importance in ligand stabilization, in
particular of Phe192 and Met193.
The results of the analysis of the crystal structures and of the
multi-trajectory MD approach coupled with free energy calcula-
tions were in agreement with the biochemical data published so far
and substantiated the random bi-bi kinetic cycle of 17b-HSD1.
Our multi-trajectory approach allowed an accurate assignment of
different enzyme conformers to each postulated step of the
catalytic cycle of 17b-HSD1. The analysis of the different
trajectories revealed an essential role for backbone and side chain
motions, in particular of the flexible bFaG9-loop, in cofactor and
substrate binding, and the existence of distinguished conformer
ensembles related to the single steps of the catalysis. Thus, it
suggested 17b-HSD1 to follow a selected-fit mechanism : E1
or NADPH binds to a transition state of the enzyme and induces
concerted conformational rearrangements of FG-segment and C-
terminal part, which then guides the enzyme along its preferred
catalytic pathway. Furthermore, the enzyme-ligand (both NADPH
and E1) motions observed in our MD study suggested a
preferential pathway for this kinetic cycle, with the cofactor
NADPH entering first, followed by the substrate E1, which then
Figure 7. Representative snapshots of the MD C3. Overlay of the representative structures of the initial position (yellow), the stable segment
(blue, step 3) and the extended part (red, step 5) of the MD C3 (1fdtB).
Kinetic Cycle of 17b-HSD1
PLoS ONE | www.plosone.org9 August 2010 | Volume 5 | Issue 8 | e12026
induces concerted enzyme adaptation to both ligands. Depending
on the presence of E1 and/or NADPH these enzyme forms
evolved into a favourable low energy conformations representative
for the specific steps, as for example observed for the ternary
complex 1I5R-NADPH-E1 (C2) where the flexible loop moved
toward the aG9-helix stabilizing both the substrate E1 and the
The small RMSD variations observed for the catalytic residues
Ser142, Tyr155 and Lys159 in all MDs suggested other regions of
the enzyme to be responsible for the proceeding of the five-step
catalytic cycle. Site-directed mutagenesis studies indicated for
His221 and Glu282 a prominent role in substrate recognition, but
not in catalysis [34,36–39]. The motions of His221 observed in the
simulations were in agreement with these results and allowed
interesting conclusions regarding the role of His221 in substrate
recognition and with respect to its chaperone function in guiding
E1 out of the SUB (Figure 7).
Ghosh et al. described two conformations for the bFaG9-loop
(‘‘substrate-entry loop’’), an opened when no steroids are present
and closed one with the steroid in the SUB [36,49]. On the
contrary, in our study we identified three main bFaG9-loop axis
and five different loop side-chains orientations, characteristic for
five steps of the bi-bi kinetic cycle. Our multi-trajectory/-complex
approach allowed us to sample a broad conformational space and
all these conformers, together with the calculated DG values,
substantiated the three cluster variant, with the enzyme in an
opened (cl1), occluded (cl3) and closed (cl2) state, depending on the
presence of steroid, cofactor-steroid or cofactor.
In conclusion, this study laid the basis not only for a better
understanding of the kinetic cycle of 17b-HSD1 and of the role of
some amino acids, in particular of Phe192, Met193, Lys195 and
Phe226, but also for possible new design strategies to inhibit 17b-
HSD1. Thus, the enzyme conformations assigned to the various
steps might represent a valid starting point for the development of
inhibitors disrupting the enzyme dynamics and thus inactivating it
by freezing it in one conformer and avoiding the cycle to evolve
further. However, direct experimental results are still needed for a
final demonstration, and, in particular, site directed mutagenesis
studies, as well as further computational investigations, are
envisaged in order to strengthen our conclusions.
Materials and Methods
1. Computational Details
Crystal structures of 17b-HSD1 were obtained from the Protein
Databank (PDB, www.pdb.org)  and further prepared using
the BIOPOLYMER module of SYBYL v8.0 (Sybyl, Tripos Inc.,
St. Louis, Missouri, USA). In detail, we used the following PDB
structures: 1a27, 1bhs, 1dht, 1equ, 1fds, 1fdtx2, 1fdu, 1fdv, 1fdw,
1i5r, 1iol, 1jtv, 1qyv, 1qyw, 1qyx, 3dhe, 3dey, 3hb4 and 3hb5. 1fdt
presents Arg37 and the loop residues Thr190 to Gly198 in two
very different conformations, denoted in this article as 1fdtA (open
state) and 1fdtB (closed state). Water molecules, sulfate ions and
the hydride inhibitor HYC (1i5r) were removed from the PDB files
and missing protein atoms were added. E2 was modified to estrone
E1 and NADP+was turned into NADPH. Hydrogen atoms and
neutral end groups were added. All basic and acidic residues were
considered protonated and deprotonated, respectively. Histidines
oriented toward the outer part of the enzyme were considered as
protonated after a prediction run made by MolProbity . For
1i5r the cofactor NADPH was merged into the enzyme after an
accurate overlay with the hybrid inhibitor HYC and the X-rays
1a27 and 1fdt. Further, every crystal structure was minimized for
500 steps with the steepest descent minimizer as implemented in
SYBYL with the backbone atoms kept at fixed positions in order to
fix close contacts, followed by 2000 steps conjugate gradient
minimization requested for an overall better starting structure.
Ligands were described with the general Amber force field
GAFF . RESP charges for estrone were calculated, while the
parameters of Ulf Ryde were taken for NADPH (charge -4)
The clustering of the crystal structures of Table 1 has been
performed by means of the CONSENSUS utility of the homology
module of MOE (www.chemcomp.com) and resulted in the two
different backbone and side-chain classifications.
2. MD simulations
Molecular dynamics (MD) and free energy computations were
carried out using Amber 9.0 suite of programs  and the
AMBER99SB force field . The simulation system was set up
using the xLeap program of the AMBER suite. The simulation
systems were surrounded by a truncated octahedral box of TIP-3
water molecules of 10 A˚ radius. Counterions were added to
neutralize the system. Prior to the free MD simulations, the
simulation systems were energy minimized for 5000 steps of
steepest descent followed by 10000 steps of conjugate gradient
optimization. The equilibration process was carried out with the
program PMEMD on the NVT ensemble using the following
procedure: the simulation system was heated during 200 ps from 0
to 200 K at constant volume conditions. This temperature was
held for additional 200 ps and afterwards raised to 300 K during
200 ps at constant volume. Protein heavy atoms, NADPH and E1
were constraint, and the force constant was gradually reduced
from 100 kcal/mol/A˚2to 2 kcal/mol/A˚2. The final coordinates of
the temperature equilibration routine were relaxed without
restraint for other 50 ps and then used for the MD production
run, performed at NPT physical conditions and without restraints.
The total simulation length differed for the various complexes
ranging from 9 to 17.5 ns. taup=2.0 and cut=10.0. Temperature
regulation was done at 1.0 atm of pressure (1 atm=101.3 kPa) by
using a Langevin thermostat with a coupling constant of
tautp=1.5 ps. The time step of the free MD simulations was
2 fs, with a cutoff of 13 A˚for the nonbonded interaction, and
SHAKE  was employed to keep all bonds involving hydrogen
atoms rigid. Electrostatic interactions were computed using the
Particle Mesh Ewald method . All simulations were carried out
in periodic boundary conditions.
The analysis of the trajectories of the MD simulations was
performed with the PTRAJ module of AMBER, the MMTSB tool
set (http://mmtsb.org)  and using visual molecular dynamics
3. Free energy calculations using the MM/PBSA method
Conventional MM-PBSA [30,59] and normal-mode (NMODE)
 calculations were performed using the AMBER 9 suite. The
electronic and Van der Waals energies were calculated by the
sander module. The polar solvation energy was calculated with the
finite-difference PB equation solver by using AMBER toolset.
For each stable sector of the MD trajectories B1–B3 and C1–
C3, longer than 4 ns, snapshots were collected every 30th frame
(every 30 ps) and used to calculate relative binding affinity (DGbind)
and absolute free energy (DG) by means of MM-PBSA methods
and NMODE. The normal mode analysis was performed to
estimate the vibrational component of the entropy. Conjugate
gradient and then Newton-Raphson minimizations until the root
mean square of the elements of gradient vector was less than
5*1025kcal/mol were carried out in absence of solvent. A
distance-dependent dielectric constant was used to mimic solvent
Kinetic Cycle of 17b-HSD1
PLoS ONE | www.plosone.org10 August 2010 | Volume 5 | Issue 8 | e12026
screening. Frequencies of the vibrational modes were computed at
300 K for these minimized structures and using a harmonic
approximation of the energies. Due to the high computational
demand, only snapshots taken every 100thframe from MD were
used to estimate -TDS.
A more detailed description of MM/PBSA and NMODE is
given in Text S2.
4. RESP charges
The ab-initio geometry optimizations for E1 was performed in
gas phase at the B3LYP/6-311+g (d,p) level of density functional
theory (DFT) by means of the Gaussian03 .
MD approach, classified as three models.
Found at: doi:10.1371/journal.pone.0012026.s001 (0.10 MB
A detailed description of the multi-trajectory/-complex
and NMODE) used.
Found at: doi:10.1371/journal.pone.0012026.s002 (0.09 MB
Additional informations to the methods (MM-PBSA
The preferred pathway is represented in red, guided by the excess
of NADPH compared to E1, and identified by the MD simulations
A1–A2, B1–B3, and C1–C3. In vitro, where the concentrations of
the reagents can be modified, different orders might also be
Found at: doi:10.1371/journal.pone.0012026.s003 (0.33 MB TIF)
Random sequential bi-bi kinetic cycle of 17b-HSD1.
bFaG9-loop, aG9-helix are highlighted. E2 and NADP+ are
rendered as sticks, whereas the enzyme as cartoons. Helices are
colored red, b-sheets in yellow and coils in green). The active site
surface is rendered as grey wireframes. (PDB entry 1a27.)
Found at: doi:10.1371/journal.pone.0012026.s004 (0.74 MB TIF)
Tertiary structure of 17b-HSD1. Rossmann fold,
HSD1. (A) 2D-scheme of the structure of 17b-HSD1; including:
Rossmann fold, cofactor binding site (COF; blue), substrate active
site (SUB; green), alternative (fused COF-SUB) active site, aG9-
helix (green ball), bFaG9-loop (yellow-red ball); gates 1 (blue), 2
(cyan) and 3 (magenta) are represented by arrows and e circles of
the same color. (B–F) Representative conformations of the 5 steps
of the catalytic cycle clustered accordingly to the side-chain
RMSD. Detailed informations about the role of the bFaG9-loop in
the various steps of the cycle are mentioned in the single charts.
The circles represent gates 1–3, and they change in size depending
whether they are opened or closed. The loop residues Phe192 (F),
Met193 (M), Glu194 (E) and Lys195 (K) are schematized
according to their effective distance from the loop axis at every
Found at: doi:10.1371/journal.pone.0012026.s005 (4.90 MB TIF)
Schematic representation of the catalytic cycle of 17b-
Different orientations of Phe192 for the 5 clusters obtained by
all-atom RMSD classification of the five loop residues (rendered in
Morphing the transition from 1fdtA to 1fdtB.
sticks, color-coded blue-violet) and for the 17 intermediate
positions (rendered in lines, magenta) obtained by simulating the
transition from 1fdtA to 1fdtB, the two extremes in the Phe192
rotation, using the Yale Morph Server (Krebs WG, Gerstein M
(2000). The morph server: a standardized system for analyzing and
visualizing macromolecular motions in a database framework.
Nucl Acids Res 28:1665–1675). A movie of this rotation is also
available (Video S1).
Found at: doi:10.1371/journal.pone.0012026.s006 (2.07 MB TIF)
A2. Time-dependent all-atom RMSD for (A) all residues of the
complexes A1 (blue - 1bhs) and A2 (red - 1fdtB), and (B) only for
the bFaG9-loop residues only. (C) Residue-dependent RMSD
fluctuation for the three complexes A1–A2.
Found at: doi:10.1371/journal.pone.0012026.s007 (3.69 MB TIF)
Analysis of the MDs of the apoform complexes A1–
Time-dependent all-atom RMSD for the complexes (A) B2 (light
blue all residues, dark blue bFaG9-loop), (B) B1 (light green all
residues, dark green bFaG9-loop) and (C) B3 (red all residues,
orange bFaG9-loop). (D) Time-dependent RMSD for NADPH of
the three complexes B2 (blue), B1 (green) and B3 (red). (E)
Residue-dependent RMSD fluctuation for the three complexes
B1–B3 (same colors as for (D)).
Found at: doi:10.1371/journal.pone.0012026.s008 (5.22 MB TIF)
Analysis of the MDs of the binary complexes B1–B3.
(A) Time-dependent all-atom RMSD for the complexes C2 (light
blue all residues, dark blue bFaG9-loop), (B) C1 (light green all
residues, dark green bFaG9-loop) and (C) C2 (red all residues,
orange bFaG9-loop). (D) Residue-dependent RMSD fluctuation
for the three complexes C1–C3.
Found at: doi:10.1371/journal.pone.0012026.s009 (4.89 MB TIF)
Analysis of the MDs of the ternary complexes C1–C3.
snapshots of the MD C1 (1fdtA-NADPH-E1), with representative
structures after (Ai) 12 ns (white), (Aii) 9 ns (yellow), (Aiii) 6 ns
(magenta) and (Aiv) 3 ns (magenta). Surfaces of the ligand binding
sites are shown in light grey. (B) Starting structure (violet) and
stable complex after 10 ns (cyan) of MD C3 (1fdtB-NADPH-E1).
Complexes are rendered in cartoon. NADPH, E1 and residues
crucial for the dynamic are shown as sticks and labeled.
Found at: doi:10.1371/journal.pone.0012026.s010 (3.91 MB TIF)
Postulated entrance mechanism for E1. (A) Four
Found at: doi:10.1371/journal.pone.0012026.s011 (2.61 MB
Morphing the transition from 1fdtA to 1fdtB.
We thank Dr. Sandrine Oberwinkler-Marchais for helpful discussion. MN
is grateful for his co-tutelle PhD between the University of Bologna and
Saarland University of which this work was a substantial part.
Conceived and designed the experiments: MN. Performed the experi-
ments: MN. Analyzed the data: MN. Wrote the paper: MN MR RWH.
Supervised MN: MR. Supervised MN.
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