J. Phys. Chem.

B 2009, 113, 945–958 945

Molecular Dynamics Simulations of the Chromophore Binding Site of Deinococcus

radiodurans Bacteriophytochrome Using New Force Field Parameters for the
Phytochromobilin Chromophore

Steve Kaminski, Grazia Daminelli, and Maria Andrea Mroginski*

Technische UniVersität Berlin, Institut für Chemie, Max-Volmer-Laboratorium, Sekr. PC 14, Strasse des 17,
Juni 135, D-10623 Berlin, Germany
ReceiVed: May 29, 2008; ReVised Manuscript ReceiVed: October 13, 2008

The conformational flexibility of the tetrapyrrolic phytochromobilin (PΦB) chromophore of the bacterio-
phytochrome Deinococcus radiodurans (DrCBD) in the Pr state has been investigated by molecular dynamics
simulations. Because these simulations require accurate force field parameters for the prosthetic group, in the
present work we developed new empirical force field parameters for the PΦB molecule that are compatible
with the CHARMM22 force field for proteins. For this reason, the new force field parameters for the nonbonded
(partial atomic charges) and bonded (bonds, angles, dihedrals, improper) energy terms were derived by
reproducing ab initio target data following the methodology used in the development of the CHARMM22
force field. This new set of parameters was employed to analyze structural and dynamical features of PΦB
inside DrCBD. The 45 ns all-atom molecular dynamics (MD) simulation reveals the existence of two stable
conformational states of the chromophore characterized by distinct torsional angles around the C-C bond at
the methine bridge connecting rings A and B of the tetrapyrrole. This result supports experimental observations
derived from NMR and resonance Raman spectroscopy. Furthermore, statistical analysis of H-bonding events
allowed us to identify (a) important H-bonds between the propionic side chains of the chromophore and the
apoprotein which may be relevant for the signal transduction step during the photoinduced cycle and (b) a
network of eight water molecules which remain in the vicinity of the chromophore during the entire 45 ns
production run.

1. Introduction mation at the methine bridges, with a nearly coplanar arrange-

Phytochromes (Phy) constitute a ubiquitous family of sensory
photoreceptors, found in plants, bacteria and fungi.1,2 These
photoreceptors regulate a variety of processes, ranging from seed
germination, formation of leaves and control of flowering time
in higher plants to phototaxis and pigmentation in bacteria. Phys
are homodimeric complexes with each polypeptide containing
three main domains: an N-terminal chromophore binding domain
(CBD) with photosensing activity, the phytochrome domain
(PHY), which is required for the formation and stability of the
physiologically active state, and the C-terminal domain which
is essential for signal transduction processes.1,2 Through a unique
interaction between the chromophore, a linear-methine bridge
tetrapyrrole (bilin), and the protein matrix (mainly the CBD and
PHY domains), phytochromes can interconvert, upon light
absorption, between two stable states: a physiologically inactive
red-absorbing (Pr) form and an active far-red-absorbing (Pfr)
form.3 The underlying molecular events controlling this process
are still not fully understood, and despite the numerous studies
in this field, the elucidation of the photointerconversion mech-
anism is still a matter of debate.
For this reason, the determination of the three-dimensional
structure of the Pr state of a bacteriophytochrome deletion
mutant from Deinococcus radiodurans is of particular impor-
tance. The 1.45 Å resolution structure of the chromophore
binding domain (DrCBD) reveals that the biliverdin chro-
mophore binds to the protein as a 2(R),3(E)-phytochromobilin
(PΦB) chromophore with a ZZZssa configuration and confor-
* Corresponding author.
10.1021/jp8047532 CCC: $40.75  2009 American Chemical Society
Published on Web 01/05/2009
ment of the A, B and C pyrrole rings (see Figure 1).4,5
Furthermore, resonance Raman spectroscopic studies on DrCBD
crystals6,7 suggest the coexistence of two or more PΦB
conformers which differ, most likely, in the torsional angles at
the A-B methine bridge. The same phenomenon has been
detected for the Agrobacterium tumefaciens phytochrome
(Agp1) crystals,6 plant phytochrome (Phy A),8 and cyanobac-
terial phytochrome in solution (Cph1).9 This structural hetero-
geneity of the bilin chromophore seems to be an intrinsic
property of phytochromes in general. Unfortunately, it cannot
be captured by crystallographic techniques. Therefore, identi-
fication and characterization of possible chromophore conform-
ers in the Pr state and their influence on the photoactivation
mechanism remains an open question.
We address this question from a computational point of view,
by analyzing the conformational fluctuations of the bilin
chromophore inside the protein binding pocket. Full-atoms
molecular dynamics (MD) simulations constitute a powerful tool
for performing this task in an efficient way. The accuracy of
this computational approach depends on the definition of the
empirical potential energy function and on the reliability of the
corresponding force field parameters. The widely used all-atom
CHARMM force field has been already successfully param-
etrized for proteins10 and can be therefore applied for modeling
the natural amino acids of the phytochrome apoprotein. The
bilin chromophore, however, does not belong to these building
blocks. To our knowledge, no empirical force field parameters
have been developed so far for linear-methine bridge tetrapyrrole
molecules. Thus, the first step toward a proper description of
946 J. Phys. Chem. B, Vol. 113, No. 4, 2009 Kaminski et al.

Figure 1. 3-D structure of DrCBD (left) and structure of model compound 2(R),3(E)-PΦB and fragmentation scheme for optimization of torsion
angle parameters (right). New atom types are written in bold letters.

the structural and dynamical properties of the entire phyto- Ab initio calculations required for the generation of target
chrome protein is the development of adequate force field data were performed with the Gaussian 03 program,14 whereas
parameters for the bilin chromophore which are compatible with molecular mechanics calculations and molecular dynamics
the already existing CHARMM all-atom force field.10,11 simulations were done using the CHARMM15 version 32b2 and
Starting from the available X-ray structure of DrCBD in the NAMD 2.616 codes.
Pr state, we employed empirical molecular dynamics to identify 2.1. Definition of the Potential Energy Function. The
and characterize possible conformers of the PΦB chromophore, CHARMM potential energy function is defined as the sum of
which may be relevant for the interpretation of the experimental various internal (bonded) and nonbonded terms in the following
data.6,7 The present paper is separated in two parts: the first way:
part (section 2) describes the development and validation of
new force field parameters for the PΦB chromophore, whereas
the second part (section 3) reports the results of molecular U(rj) ) ∑ Kb(b - b0)2 + ∑ KUB(S - S0)2 +
dynamic simulation of DrCBD in solution. Special emphasis is bonds UB
given in elucidating the conformational flexibility of the
chromophore and the stability of the hydrogen bond and water
∑ Kθ(θ - θ0)2 + ∑ Kχ(1 + cos(nχ - δ)) +

[( ) ( ) ]
angle dihedral
network in the protein binding pocket during the MD simulation. Rij 12 Rij 6
∑ Kφ(φ - φ0)2 + ∑ εij
rij
-2
rij
+
improper nonbond
2. Development and Validation of Force Field qiqj
Parameters (1)
4πεrij
The optimization and evaluation of new force field parameters
for phytochromobilin was performed following the iterative
procedure used for developing the CHARMM22 force field.10 where K denotes force constants for bonds (b), Urey-Bradley
This procedure is based on the reproduction of target data at a 1,3-distances (S), bond angles (θ), dihedral angles (χ) and
molecular mechanics level. The target data include structural improper dihedral angles (φ) and b0, S0, θ0, χ0 and φ0 are the
data, energies (e.g., rotational barriers) and intermolecular corresponding equilibrium values of the individual terms. The
interaction energies of model compounds estimated from ab nonbonded interactions are described as the sum of Lennard-
initio calculations.10,12,13The model compounds are simply Jones 6-12 and Coulomb terms. The first depends on the
isolated molecules containing the structural units (bond lengths, Lennard-Jones parameters, εij and Rij, for the atom pair i and j
bond angles and dihedral angles) associated with those param- and the distance rij between them. The Coulomb terms are a
eters that are to be optimized. The parametrization strategy is function of the atomic partial charges qi and qj and their relative
then to maximize the agreement between the target data and distance rij.
the corresponding molecular properties of the model compounds 2.2. Model Compounds. The model compound for the
computed using the empirical force field. protein-bound chromophore consists of an isolated protonated
The use of this parametrization approach guaranties consis- 2(R),3(E)-phytochromobilin (PΦB) molecule in a ZZZssa
tency with the available force fields CHARMM2210 and conformation/configuration of the methine bridges as found in
CHARMM2711 employed to model the phytochrome apoprotein. the X-ray structure of DrCBD shown in Figure 1. The propionic
Details about the parametrization procedure are described in side chains were protonated (neutral) and the linkage of the
the paragraphs below. chromophore to the protein was saturated by a hydrogen atom.
MD Simulations of the Chromophore Binding Site of DrCBD J. Phys. Chem. B, Vol. 113, No. 4, 2009 947

TABLE 1: Atom Types, Labels and Optimized Partial Charges of the PΦB Moleculea
atom atom type partial charge atom atom type partial charge atom atom type partial charge
CAC CPM -0.082 C3B CPY6 -0.091 HO3 HA 0.09
HAC HA 0.123 O_B O -0.407 HO4 HA 0.09
C1C C 0.289 CHB CPY2 -0.448 CGD CC 0.62
H2C HA 0.057 HHB HA 0.219 O2D OC -0.76
N_C NR1 -0.555 CAB CE1 -0.332 O1D OC -0.76
H_C H 0.332 HAB HE1 0.307 CBA CT2 -0.28
C4C CA 0.312 CBB CE2 -0.269 HO1 HA 0.09
C3C CPY1 -0.160 HV1 HE1 0.189 HO2 HA 0.09
C2C CT1 0.315 HV2 HE2 0.153 CGA CC 0.62
O_C O -0.445 CBC CT2 0.043 O2A OC -0.76
CHD CPY3 -0.501 HL1 HA 0.058 O1A OC -0.76
HHD HA 0.214 HL2 HA 0.058 CAA CT2 -0.18
C1D CPA 0.375 CMC CT3 -0.119 HO5 HA 0.09
N_D NR1 -0.543 HE1 HA 0.038 HO6 HA 0.09
H_D H 0.306 HE2 HA 0.038 CAD CT2 -0.18
C4D CPA 0.364 HE3 HA 0.038 HO7 HA 0.09
C3D CPB -0.091 CMD CT3 -0.024 HO8 HA 0.09
C2D CPB -0.319 HD1 HA 0.022
C1A CPA 0.119 HD2 HA 0.022
N_A NR1 -0.654 HD3 HA 0.022
H_A H 0.355 CMA CT3 -0.075
C2A CPY4 -0.038 HA1 HA 0.050
C3A CPB -0.155 HA2 HA 0.050
C4A CPA 0.487 HA3 HA 0.050
C4B C 0.476 CMB CT3 -0.018
N_B NR1 -0.560 HB1 HA 0.049
H_B H 0.425 HB2 HA 0.049
C1B CA 0.452 HB3 HA 0.049
C2B CPY5 -0.115 CBD CT2 -0.28
a
Values related to the propionate chains are written in italic letters. New atom types are in bold letters.

To account for electron delocalization effects, the entire PΦB
molecule was used for the optimization of the internal bond
lengths, bond angles and improper dihedral parameters and the
nonbonded electrostatic parameters. Figure 1 (right) shows the
chromophore structure together with the atomic labels for
the heavy atoms. The list of all atoms belonging to the PΦB
chromophore and their corresponding atom type is given in
Table 1. Six new atom types were specifically created for the
PΦB molecule to be able to define new torsion potentials and
consequently improve the accordance with the internal properties
of the target structure, as will be shown later.
For the evaluation of torsion parameters for the chromophore,
the PΦB molecule was divided into smaller fragments (see
Figure 1 (right)) to reduce computational cost. The hexameth-
ylpyrromethene (HMPM) and pentamethylpyrrolone (PMPO)
molecules were used to model the torsions at the methine bridge
between inner rings B and C and the methine bridges between
inner rings B/C and the outer ring A/D, respectively. The torsion
parameters at the chromophore-protein linkage site were evalu-
ated using the [(aminosulfanyl)oxo]pyrrolidine acid (ASOP) and
for the parameter associated with the vinyl function at ring D
we used a methylenevinylpyrrolone (MVPO) molecule a as
model compound. In this way all torsion angle parameters which
are relevant for the chromophore flexibility were accurately and
efficiently determined.
2.3. Target Data. The target data for optimizing internal
force field parameters such as bond, angle and improper torsion
parameters, was derived from the DFT optimized geometries
of the entire PΦB chromophore. The same structure has been
used for the evaluation of partial charge. The use of a complete
PΦB molecule as the target structure guaranties the correct
description of the electron delocalization effects. However, to
reduce computational cost, the dihedral parameters were de-
veloped using the molecular fragments mentioned in the
previous section. All ab initio calculations were performed in
vacuo using Becke’s three parameters functional (B3LYP)18 and
the 6-31G(d) basis set.
2.4. Nonbonded Parameter. The optimization of nonbonded
force field parameters involves electrostatic and van der Waals
interactions. The main goal is to obtain a set of parameters that
can reproduce, in a reliable way, the water-solute interaction,
where water is described through a TIP3P model.17
Following MacKerell’s procedure,10 the partial atomic charges
for the PΦB chromophore were obtained by computing, at a
quantum mechanical level, minimum interaction energies and
distances between the model compound and water molecules.
We performed the calculations using fourteen water molecules
located at various sites as shown in Figure 2. The PΦB geometry
was previously optimized in vacuo using the hybrid density
functional method B3LYP18 with the 6-31G(d) basis set whereas
the water molecules were built according to the TIP3P water
model.17 The minimum interaction distances were estimated
through single point energy calculations of the various
PΦB-water complexes at the HF/6-31G(d) level of theory.
These complexes were built by systematically varying the
interaction distances in steps of 0.01 Å. Interaction energies
were then estimated by subtracting the sum of the monomer
energies from the energy of the entire complex. Because the
chromophore is a charged compound, the target HF/6-31G(d)
interaction energies where not scaled.10 Initial partial atomic
charges were obtained from fitting the electrostatic potential
using the CHELPG19 method implemented in Gaussian03 while
simultaneously reproducing the overall dipole moment of the
chromophore.
The partial charges of atoms belonging to the propionate side
chains were not optimized. These values were taken form the
already existing parameters for the heme cofactor20 without
further adjustment.
948 J. Phys. Chem. B, Vol. 113, No. 4, 2009 Kaminski et al.

TABLE 2: Optimized Parameters for Newly Defined

Dihedral Angles of the PΦB Chromophore
dihedral parameter Kχ (kcal/mol) n χ0 (deg)
Linking Fragment 1 (ASOP)
CT2-SE-CT2-CPM 0.7 3 10.0
CT2-SE-CT2-CPM 1.1 1 88.0
Linking Fragment 2 (ASOP)
SE-CT2-CPM-CPY1 1.4 2 45
Linking Fragment 3 (ASOP)
CT2-CPM-CPY1-CA 2.4 2 180.0
CT2-CPM-CPY1-CA 16.4 1 4.0
Double Bond Pyrrolinone Fragment Ring C-D (PMPO)
CPA-CPY2-CA-CPY5 11.8 1 0.0
CPA-CPY2-CA-CPY5 3.0 2 190.0
Single Bond Pyrrolinone Fragment Ring C-D (PMPO)
CPB-CPA-CPY2-CA 2.0 3 0.0
Figure 2. Relative orientations of fourteen water molecules with CPB-CPA-CPY2-CA 4.4 2 138.0
respect to the PΦB molecule for the evaluation of partial charges. CPB-CPA-CPY2--CA 2.1 1 243.0
The partial atomic charges obtained for the PΦB molecule Pyrrol Fragment 1 (HMPM)
are listed in Table 1. CPY4-CPA-CPM-CPA 9.2 2 183.0
CPY4-CPA-CPM-CPA 4.5 1 2.0
The quality of the optimized partial charges was estimated
CPY4-CPA-CPM-CPA 2.2 3 190.0
by reproducing minimum interaction energies and water-
chromophore distances derived at the ab initio level of theory Pyrrol Fragment 2 (HMPM)
CPB-CPA-CPM-CPA 8.9 1 184.0
(see Table 4). Distances were deliberately underestimated in
CPB-CPA-CPM-CPA 1.5 2 0.0
about 0.2 Å compared to the target data following the established CPB-CPA-CPM-CPA 1.9 3 196.0
parametrization procedure.10 Because an accurate reproduction
Double Bond Pyrrolinone Fragment Ring A-B (PMPO)
of both interaction energies and distances was impossible we
CPY1-CA-CPY3-CPA 1.9 1 0.0
always tried to find in all cases a good compromise between CPY1-CA-CPY3-CPA 6.4 2 178.0
them. CPY1-CA-CPY3-CPA 1.8 3 160.0
As an additional criterion for evaluating the quality of the
Single Bond Pyrrolinone Fragment Ring A-B (PMPO)
partial charges we simultaneously tried to reproduce the overall CA-CPY3-CPA-CPB 1.2 3 62.0
dipole moment. To account for the missing explicit inclusion CA-CPY3-CPA-CPB 4.4 2 162.0
of polarizability in classical force fields, the dipole moment CA-CPY3-CPA-CPB 2.5 1 252.0
should be overestimated10 to a certain extent compared to the Vinyl Fragment (MVPO)
values obtained with QM calculations. Therefore, to be con- CPY5-CPY6-CE1-CE2 0.9 1 0.0
sistent with CHARMM force field parameters for proteins, the CPY5-CPY6-CE1-CE2 2.35 2 180.0
partial charges of the chromophore were adjusted so as to obtain CPY5-CPY6-CE1-CE2 0.5 3 0.0
an empirical dipole moment 10% larger than the ab initio target
values. Empirical- and QM-calculated dipole moments as well
as interaction energies and distances for the PΦB molecule are The target data consists of the DFT optimized geometries of
given in Table 4. the model compound (for evaluation of bond, angle and
Lennard-Jones parameters
ij and Rij were calculated by means improper torsion parameters) and of rotational energy profiles
of the Lorentz-Berthelot mixing rules using the CHARMM27- of its fragments (for the evaluation of dihedral torsion param-
Lennard-Jones parameters of atoms types. Because these atomic- eters). The quantum mechanical geometry optimizations were
Lennard-Jones parameters are transferable between atom types carried out under tight convergence criteria to a final gradient
which are chemically similar, those parameters required for the of 10-5 au, using the X-ray crystallographic structure as input
PΦB chromophore were simply collected from other compounds geometry.
such as heme. Therefore, in this work we did not optimize any All geometry optimizations in CHARMM involving the entire
Lennard-Jones atomic parameters for PΦB. chromophore and fragments were performed using the adopted
2.5. Internal Parameters. The parametrization of the internal basis Newton-Raphson method (ABNR) followed by several
force field parameters of PΦB was done iteratively starting from steps using the Newton-Raphson (NRAPH) minimizer.
a set of reasonable initial values from already parametrized To ensure a reliable conformational behavior of the chro-
analogue structures. Most of the initial internal parameters (bond, mophore, ten new harmonic torsion potentials were defined (see
angles, dihedral, improper) and atom types correspond to the Table 2) to describe rotations at the methine bridges, the linkage
CHARMM parameters previously optimized for the heme to the protein (CYS 24) and at the vinyl functionality on ring
molecule20 (toppar_all22_prot_heme.str). Among all internal D. The corresponding torsional force field parameters were
force field parameters of the PΦB chromophore defined in eq determined by fitting the torsional profiles of the various
1, in the present work we optimized the corresponding equi- molecules (Figure 3) obtained by quantum mechanical calcula-
librium bond lengths (b0), bond angles (θ0), improper (φ0) and tions with the corresponding molecular mechanics potential
dihedral angles (χ0) as well as harmonic force constant Kφ, Kχ energy function. This function was constructed according to eq
and periodicity and phase parameter (n, δ) for the dihedral 1 and employing, in a first stage, the initial internal parameters
torsions. The harmonic force constants Kb, KS, Kθ together with chosen for the entire PΦB molecule and the corresponding point
the all parameters of the propionate side chain were kept fixed charges calculated as described in section 2.4. The QM and MM
during the optimization procedure. potential energy curves were scanned in steps of 10° by
MD Simulations of the Chromophore Binding Site of DrCBD J. Phys. Chem. B, Vol. 113, No. 4, 2009 949

Figure 3. Potential energy surfaces for partially constrained model compounds. The surfaces were scanned in steps of 10°. The black curves
denote the target data derived using ab initio methods (DFT/B3LYP/6-31G(d)), and the gray dashed-curves result from optimized torsion parameters
(see Table 2). Potential curves were shifted by setting the lowest energy conformations for each curve equal to zero.

performing constraint energy minimizations with a rms force analogue molecular mechanics calculations. In each step only
criterion of 10-4 for the ab initio calculations and 10-6 for the torsional angle in question was kept fixed to a certain value
950 J. Phys. Chem. B, Vol. 113, No. 4, 2009 Kaminski et al.

TABLE 3: Optimized Parameters for All Newly Defined Internal Coordinates (Except Torsions) of PΦB
bond Kb b0 bond Kb b0
parameter [(kcal/mol)/Å2] (Å) parameter [(kcal/mol)/Å2] (Å)
NR1-CPA 377.2 1.3817 CPY3-CA 360.0 1.3676
NR1-C 260.0 1.4190 CPY3-CPA 360.0 1.4225
CT2-CPM 230.0 1.4830 CPY4-CPA 299.8 1.4052
NR1-CA 377.2 1.4117 CPY4-CPB 340.7 1.4018
CPY1-CA 305.0 1.4772 CT2-CPY4 230.0 1.4946
CT1-CPY1 230.0 1.4907 CPY5-CA 305.0 1.4620
CPM-CPY1 360.0 1.3266 CT3-CPY5 230.0 1.4850
CPY2-CA 360.0 1.3800 CPY6-C 250.0 1.4711
HA-CPY2 367.6 1.0902 CPY6-CE1 450.0 1.4290
CPY2-CPA 360.0 1.4455 CPY6-CPY5 305.0 1.3470
angle Kθ θ0 angle Kθ θ0
parameter [(kcal/mol)/rad2] (deg) parameter [(kcal/mol)/rad2] (deg)
NR1-CPA-CPB 122.0 110.0 CPY1-CA-CPY3 61.6 127.57
CPA-NR1-CPA 139.3 116.3 CA-CPY3-CPA 94.2 122.80
CPM-CPA-NR1 88.0 131.8 CPY3-CPA-CPB 61.6 125.07
NR1-C-O 80.0 118.0 CPM-CPA-CPY4 61.6 132.50
HA-CT2-CPM 49.3 107.5 CPB-CPY4-CPA 30.8 136.01
C-NR1-CA 50.0 133.5 CPY4-CPB-CPA 30.8 145.01
HA-CPM-CPY1 12.7 117.44 CT2-CT2-CPY4 70.0 114.7
CT2-CPM-CPY1 45.8 117.49 NR1-CA-CPY5 122.0 112.4
NR1-CA-CPY1 122.0 115.0 CPY5-CA-CPY2 61.60 124.97
CT1-CPY1-CPM 45.8 116.6 CT3-CPY5-CA 45.8 115.9
CT1-CPY1-CA 45.8 128.0 NR1-C-CPY6 20.0 109.5
CPY1-CT1-C 52.0 113.7 C-CPY6-CPY5 52.0 116.0
CPM-CPY1-CA 61.6 124.1 C-CPY6-CE1 70.0 114.49
CPY2-CPA-NR1 88.0 112.39 CE2-CE1-CPY6 40.0 117.6
CPY2-CA-NR1 88.0 129.0 CA-CPY5-CPY6 40.0 116.5
CA-CPY2-CPA 94.2 127.0
improper Kφ φ0 improper Kφ φ0
parameter [(kcal/mol)/rad2] (deg) parameter [(kcal/mol)/rad2] (deg)
CPA-CPB-NR1-CPM 200.0 0.0 CPA-CPM-NR1CPY4 61.0 0.0
CPA-CPB-NR1CPY2 140.0 0.0 CPY4-CPA-CPB-CT2 64.0 0.0
CPA-CPB-NR1CPY3 145.0 0.0 CPY5-NR1-CA-CPY2 7.0 180.0
CPY1-NR1-CACPY3 140.0 180.0 CPY6-CE1-C-CPY5 180.0 0.0
while the rest of the structure was allowed to relax. Because of heights of rotational barrier as well as the overall shape of the
the chemical similarity between the outer rings A-B and C-D potential surfaces (Figure 3). Most problematic in this respect
(see Figure 3C) one set of target data (here the structure of ring was the potential describing single bond rotations of the linking
C-D was chosen) was sufficient to model the corresponding fragment to the protein (Figure 3f,g), because of the high
methine bridge torsions. Due to electron conjugation at the flexibility of this structure. Because we are not interested in
central methine bridge, the ab initio potentials for the two bond predicting Z/E isomerization energy barriers at double bonds,
torsions are practically identical. only a certain region of the potential energy curve around a
We now can summarize the iterative procedure for the local minimum was scanned. Within these limited dihedral-angle
parametrization of the nonbonded parameters. At first, partial intervals the torsion potentials were in general very well
charges and internal parameters (no torsions) have been derived reproduced. However, for the rotation around the CdC bond
for the entire chromophore. These parameters have been at the AB methine bridge (Figure 3a) although the overall
transferred to the smaller fragments to evaluate torsion param- curvature and local minimum at around 195° could be well
eters that were transferred back to the chromophore to refine predicted, the energy barrier and the second local minimum
the residual internal parameters and partial charges until self- slightly differ from the QM target.
consistency within all force field parameters for the chromophore 2.6. Validation of Force Field Parameters. Validation of
was achieved. The optimized bond length, bond angle and the final set of all internal and nonbonded parameters optimized
improper torsion parameters for the protein bound chromophore for the PΦB cofactor was done by comparing the energy
are listed in Table 3 whereas the corresponding optimized minimized structures of the isolated PΦB molecule and the
dihedral parameters are given in Table 2. model fragments (see section 2.2) obtained at the QM and MM
Furthermore, it is important to mention that, for the MM in levels. Energy minimizations of the PΦB molecule were
vacuo calculations, the force field parameters of the PΦB- performed starting from the crystal structure (the protein-bound
propionic side chain in its neutral form were taken from the PΦB in DrCBD). To avoid the formation of hydrogen bonds
acetic acid included in the CHARMM force field (see between the propionate side chains and the PΦB backbone
toppar_all22_prot_model.str/RESI ACEH). In the condensed during minimizations in vacuo, which may distort the validation
phase, molecular dynamics simulations of PΦB with charged of the new force field parameters, the PΦB-acidic groups were
propionate side chain were performed using the force field removed.
parameters derived for the heme cofactor. Figure 4 (bottom) shows the resulting MM- and QM-
During the parametrization of the dihedral torsions emphasis minimized structure of PΦB and the corresponding root-mean-
was placed on the accurate reproduction of the local minima, square deviations (rmsd) for all heavy atoms after optimal
MD Simulations of the Chromophore Binding Site of DrCBD J. Phys. Chem. B, Vol. 113, No. 4, 2009 951

TABLE 4: Interaction Energies and Distances for Chromophore-Water Complexes Evaluated at the ab Initio (HF/6-31G(d))
and Empirical Levels
HF/6-31G(d) empirical/CHARMM deviations
interaction
Emin (kcal/mol) Rmin (Å) Emin (kcal/mol) Rmin (Å) ∆E (kcal/mol) ∆R (Å)
1 -2.549 3.39 -2.762 3.38 -0.213 -0.01
2 -3.789 3.27 -3.883 3.43 -0.094 0.16
3 -5.418 2.63 -5.386 2.58 0.032 -0.05
4 -6.154 2.60 -6.036 2.55 0.118 -0.05
5 -3.059 3.39 -3.0427 3.39 0.016 0.0
6 -3.095 4.09 -4.479 3.40 -1.384 -0.69
7 -2.329 3.33 -2.497 3.33 -0.168 0.0
8 -2.672 3.80 -2.583 3.51 0.082 -0.29
9 -2.909 2.06 -3.887 1.82 -0.98 -0.24
10 -3.692 2.61 -4.298 2.54 -0.61 -0.07
11 -2.450 3.41 -2.597 3.34 -0.147 -0.07
12 -0.739 3.25 -0.977 3.26 -0.238 0.01
13 -5.489 2.51 -5.648 2.36 -0.159 -0.15
14 -4.966 3.20 -5.019 3.14 -0.053 -0.06
average differential energy: 0.307 kcal/mol
average difference distance: 0.184 Å
dipole moment (Debye)/HF-6-31G(d): total ) 13.370, x ) -13.369, y ) 0.104, z ) -0.162
dipole moment (Debye)/empirical: total ) 14.646, x ) -14.556, y ) -1.426, z ) 0.772

the torsional angles toward all other internal and nonbonded

parameters. The average deviation for all 15 defined improper
angles is 0.665°, which is again satisfactory.
In addition to the PΦB chromophore, we evaluated the overall
structural agreement between the QM and MM optimized
structures of the small model fragments shown in Figure 4. As
starting structures for the unconstrained geometry optimizations
we chose geometries of the fragments lying in the vicinity of
important conformational local minima. These unconstrained
geometry minimizations we performed using the same condi-
tions as described above for minimizations of the entire
chromophore. Taking as a reference the equilibrium structure
of PΦB in the Pr state, we chose a trans-like conformation of
the fragment mimicking rings C and D and a cis-like conforma-
tion of the fragments involving rings A and B as well as rings
B and C. For the fragment describing the attachment of the
cofactor to the protein we used a starting geometry where all
three considered torsion angles were near their local minima.
Concerning the fragment with the vinyl functionality, both cis-
and trans-conformations were taken into account. The resulting
rms deviations between the optimized QM and MM structures
listed in Figure 4 are satisfactory.

3. Molecular Dynamics Simulations

Figure 4. Optimized ab initio (DFT/B3LYP/6-31g(d)) (dark gray) and
empirical (light gray) structures of the PΦB chromophore without
propionate chains and the small model fragments used in the param-
etrization proccedure. rmsd are given for non-hydrogen atoms after
optimal superposition.
superposition of the two structures. In the case of the PΦB
molecule there is an overall very good agreement between the
MM-optimized and the ab initio structures as indicated by the
rmsd of only 0.282 Å. The average deviation for 39 bond lengths
between QM and MM optimized structure including all heavy
atoms is about 0.009 Å, which is satisfactory for our needs as
well as the deviation of 0.941° for 40 bond angles. Significantly
larger is the average difference of 3.393° involving all methine
bridge, vinyl and link-fragment torsion angles. This large
deviation can be explained in terms of a higher sensitivity of
3.1. Computational Details. To check the performance of
the new force field parameters for PΦB in a protein environ-
ment, we performed molecular dynamics simulations of the
entire CBD from Drph1 phytochrome photoreceptor. Initial
coordinates for the protein were taken from the Protein Data
Bank (reference: 2O9C/conformation A/resolution: 1.45 Å).
Missing heavy atoms of several residue side chains (ASP 4,
ARG 70, GLU 193, HIS 196, ARG 310) were added using the
CHARMM software, subsequently hydrogen atoms were in-
corporated to the structure by means of the HBUILD routine.21
The assignment of a proton to titratable groups was done on
the basis of visual inspection of the environment of the charged
amino acids and histidine residues. In this way, all lysine (2)
and arginine (19) residues found in the crystal structure were
protonated and all aspartate (11) and glutamate residues (19)
were considered to be deprotonated. His 260 and His 290 were
modeled with a hydrogen at the δ-nitrogen whereas the
remaining histidine residues were protonated at the
-nitrogen.
952 J. Phys. Chem. B, Vol. 113, No. 4, 2009
Figure 5. Root-mean-square deviations (rmsd) for the protein (light gray), its backbone atoms (dark gray) and the chromophore heavy atoms
(black) and as a function of time.
This gives a strongly negatively charged protein with a total
charge of -10 e.
Afterward, the protein was solvated in a cubic box of water
with an initial volume of 884736 Å3 (26307 solvent water
molecules). The shortest initial distance between protein and
water box walls was approximately 15 Å. This distance is
deliberately larger than the chosen van der Waals long-range
cutoff (12 Å) to avoid long-range interactions between the
protein and its images. The TIP3P potential was used for the
water molecules.17 The protein charge was neutralized by
inserting chloride and sodium counter-ions in the following
manner: each charged residue not involved in salt bridges was
individually neutralized by placing a counterion within a 3 Å
radius from it, whereas the remaining ions were placed randomly
in the water box.
All subsequent calculations concerning heating, equilibration
and production were performed with the NAMD program
(version 2.6)16 using the CHARMM27 force field. At first the
solvent waters were heated to 300 K and equilibrated for 40 ps
while keeping all other parts of the system fixed. Subsequently,
several energy minimization runs (2000 steps of conjugate
gradient) were performed in which the initial harmonic restraints
(10 kcal/(mol Å2)) applied to the chromophore and all heavy
backbone atoms of the protein were gradually released until
the entire system was free. These harmonic positional restrains
were described with a quadratic potential energy function.
Afterward, the system was heated up to 300 K during 60 ps
using Langevin dynamics with restraint chromophore and
protein backbone atoms. These steps were followed by 100 ps
molecular dynamics simulation under constant pressure, constant
temperature (NPT) conditions using a combination of the
Langevin Piston Nose-Hoover method,22,23 as implemented in
NAMD. Here again, the harmonic quadratic restraints were
stepwise released until the system was totally unrestrained.
After the system was equilibrated, we proceeded with the
production run consisting in 50 ns molecular dynamics simula-
tion using a reduced Langevin damping factor (varying from
5.0 to 1.0) to more closely approximate free dynamics (NPT
conditions at 300 K). All simulations were done under periodic
boundary conditions with the long-range electrostatic interaction
described via the particle-mesh-Ewald method.24 For the van
der Waals interactions a cutoff (12 Å) was used in combination
with a switching function. To use a 2 fs time step, all bond
lengths between heavy atoms and hydrogen have been con-
Kaminski et al.
strained to their minimum energy values by applying the
SHAKE algorithm.25 All molecular structures were drawn using
the visual Molecular Dynamics Software VMD 1.8.6.26
3.2. Dynamic Properties of the Protein. In Figure 5 the
root-mean-square deviations (rmsd) for all heavy atoms of the
protein and chromophore were plotted over the simulation time
using the first frame of the simulation as the reference struc-
ture (conformations were stored each 1 ps). Because the rmsd
increased progressively during the first 5 ns of the MD
simulation, they were considered as part of the equilibration
phase and were not used for further analysis. The rmsd during
the remaining 45 ns of the simulation, on the other hand,
remained stable and were therefore used for the statistical
evaluation of the trajectory. The average of the rmsd evaluated
over the 45 ns of the production run yield 2.4 Å for the entire
protein, 1.9 Å for the protein backbone and 1.3 Å for the
chromophore (excluding hydrogen atoms). These values are
similar to those previously calculated for cytochrome c oxi-
dase.13 The slightly higher average rmsd for the protein results
from the larger mobility of the protein side chains. During the
simulation the overall shape of the protein is conserved as
indicated by the small fluctuations (0.12 Å) of its calculated
average radius of gyration of 20.33 Å. The rmsd involving all
heavy atoms of the average protein structure resulting from the
MD simulation and the crystal structure of DrCDB is 1.82 Å.
The mayor structural differences between these two structures
are found in the unstructured coil region near the N-terminus
(residues 4-11) as well as in the three R helices defined by the
residue sequences 58-62, 66-69 and 81-88. Structurally very
conserved on the other hand, are the six β strands surrounding
the chromophore.
To analyze the mobility of the molecular system, the atomic
root-mean-square fluctuations (rmsf) were calculated from the
MD simulation. Under the assumption of isotropic atomic
fluctuations, the rmsf are related to the experimental B-factors
(Debye-Waller or temperature factor) from the X-ray structure
via the following expression27
3
〈∆ri2〉 ) Bi (2)
8π2
where Bi and the square root of 〈∆ri2〉 represent the temperature
factor and the root-mean-square fluctuations associated with
MD Simulations of the Chromophore Binding Site of DrCBD
Figure 6. Per residue averaged root-mean-square fluctuations over all atoms derived from of experimental B-factor (black) and calculated from
MD simulation (red).
atom i, respectively. The 〈∆ri2〉 of each atom was calculated
with respect to its average position during the MD simulation.
Figure 6 shows the experimental and the calculated rmsf
averaged over the individual amino acids. The theoretical values
are systematically larger than the experimental ones by almost
a factor of 2. Therefore, one should compare these values in a
qualitative manner28 to determine whether the relative mobilities
predicted by the MD simulation for different regions of the
system are in accordance with the experiment. In fact, the results
plotted in Figure 6 indicate that for DrCBD that is, in general,
the case. For example, we see that for one of the most flexible
parts in the protein, a 3-10 helix (residues 130-134), simula-
tion and experiment are in a qualitative good agreement.
However, large deviation in flexibility can be observed for the
loop region between two β-strands comprising residues 279-282
as well as for the loop region between residues 104-108. In
particular, the predicted mobility of residues lying in the vicinity
of the chromophore, such as CYS24, ASP207, MET 259, HIS
260, SER 272, SER 274 and HIS290, agrees very well with
the corresponding B-factors.
3.3. Dynamic Properties of the Chromophore. When
analyzing the 45 ns production run of the MD trajectory, we
noticed a particularly interesting behavior of the chromophore
and its interaction with residue HIS 260. During the simulation,
the system seems to jump between two states characterized by
the presence (state 1) or absence (state 2) of a water molecule
localized between rings B and C, the so-called pyrrole water
illustrated in Figure 11. In state 1, the pyrrole water is hydrogen
bonded to HIS260 and to the NH group on ring A as observed
in the crystal structure. In state 2, on the other hand, this pyrrole
water moves away from the binding pocket and HIS260 comes
closer to ring A strengthening its interaction with the chro-
mophore. Each state is therefore characterized by distinct N_C
(ring A in PΦB)-ND1 (H260) distances as shown in Figure 7.
To evaluate in more detail the structural properties of the
chromophore in each of these states, average structures were
J. Phys. Chem. B, Vol. 113, No. 4, 2009 953
calculated over the time intervals (each 4 ns long) indicated in
Figure 7 with dashed boxes. The results are presented in Figure
8, where the average structures for state 1 (left) and for state 2
(right) are superimposed with the crystallographic structure. For
state 1, we obtain an overall good agreement concerning the
chromophore and linkage to the protein (rmsd for PΦB of 0.58
Å). The distance between ring A of the chromophore and HIS
260 (5.23 Å) is comparable to the X-ray structure (4.99 Å).
The average structure of state 2 reveals, on the other hand,
significant distortions of the chromophore structure (rmsd for
PΦB: 0.92 Å) that mainly involve ring A and ring C. Unlike
the crystal structure, ring A moves out of the plane formed by
the two coplanar inner rings. The dihedral angles at the A-B
methine bridge of the average structures predicted for these two
chromophore states are 13.41° (single bond)/18.44° (double
bond) in state 1 and 18.48° (single bond)/17.21° (double bond)
in state 2. These torsional angles are significantly larger
compared to those measured in the X-ray structure: 7.55° (single
bond)/6.773° (double bond). In addition, from an energetic point
of view, we cannot distinguish one state from the other. The
energy plots along the simulation show that the protein and the
chromophore remain stable. This would be in agreement with
the fact that at room temperature and during 45 ns simulation
both states are almost equally occupied (see Figure 7). Other
regions of the chromophore are structurally similar for both
states, although slight deviations also exist in the vicinity of
ring D.
The conformational flexibility of the chromophore was further
investigated by analyzing the statistical distribution of all
dihedral angles at the methine bridges. Except for the single
bond torsion between rings A and B (A-B methine bridge) all
distributions could be perfectly fitted with a single Gaussian
function with widths oscillating between 8° to 10°. As an
example we plotted in Figure 9 (bottom, left) the statistical
distribution of the dihedral angle at the double bond of the A-B
methine bridge. In this case a single Gaussian function with
954 J. Phys. Chem. B, Vol. 113, No. 4, 2009 Kaminski et al.

Figure 7. Time dependent distance between ring A (N_C) of the chromophore and HIS 260 (ND1). Dashed boxes indicate the time interval from
which average structures of state 1 and state 2 were determined.

Figure 8. Chromophore binding pocket: Crystallographic structure (dark gray) superimposed with average structures (light gray) resulting from a
4 ns MD simulation of state 1 (left) and state 2 (right). rmsd values for the heavy atoms of PΦB are 0.58 Å (state 1) and 0.92 Å (state 2).

maximum at 18.7° and a bandwidth of 14° is sufficient for a
satisfactory description of the dihedral angle distribution. The
distribution of the A-B torsion around the single bond,
however, displays an asymmetric shape (see Figure 9 bottom
right), which can only be fitted if at least two Gaussian
components are considered. The maxima of these two compo-
nents are located at 9.5° and 16.2° with bandwidths of 12° and
9°, respectively, which are slightly larger than those required
for fitting the angular distribution of the other torsions. Because
the band widths of the distribution curves reflect the degree of
fluctuation associated with each torsional angle, we conclude
that the A-B methine bridge is more flexible than the B-C and
C-D methine bridges. This conclusion is in agreement with the
experimental X-ray B-factors for the chromophore which are
larger for ring A than for the rest of the PΦB chromophore. In
addition, these results would also support experimental observa-
tions derived from NMR9 and resonance Raman spectroscopy.6
By plotting the N_C-ND1(HIS260) distance as a function
of the dihedral angles at the A-B methine bridge (Figure 9, top
left and right) we could extract some information about how
these quantities correlate. The scatterplots for the two dihedral
angles show basically two cluster of points located at the average
N_C-ND1 distances characterizing the protein’s state 1 and
state 2 described above. In this respect, the upper cluster
corresponds to state 1 whereas the lower cluster stands for state
2. For the scatter plot associated with the double bond torsion
MD Simulations of the Chromophore Binding Site of DrCBD J. Phys. Chem. B, Vol. 113, No. 4, 2009 955

Figure 9. Dihedral angle distributions (bottom) for the double (left) and single (right) bond torsion between rings A and B estimated over the 45
ns MD simulation. Scatter plots (top) show the correlation between these dihedral angles and the N_C (ring A)-ND1 (HIS 260) distance distribution.

(Figure 9 top left), both clusters of points are arranged along a

line corresponding to the maximum of the Gaussian curve
describing the distribution of double-bond dihedral angle at the
A-B methine bridge. This indicates that the fluctuations at
the A-B double bond torsions are practically not affected by
the position of the HIS260 residue. The opposite is found for
the A-B single-bond torsion. In this case, in the corresponding
scatterplot (Figure 9 top right) the upper points cluster is not
only more dispersed than the lower one but also shifted to lower
angles, suggesting that the position of the HIS260 influences
the average value and fluctuations of the A-B single bond
torsion. More precisely, we see that in state 2 (short distance)
the torsional angle at the A-B methine bridge is larger than
that for state 1 (large distance), indicating that the displacement
of HIS60 toward the chromophore brings ring A out of the plane
formed by rings B and C. In fact, a N-H · · · N hydrogen bond
is built between HIS260 and ring A in PΦB. The presence of
the pyrrole water in the protein-binding pocket (state 1) on the
other hand, allows for a larger flexibility of the A-B methine
bridge.
Summarizing, the simulation predict two stable states for the
PΦB chromophore in the binding pocket characterized by (a) Figure 10. Hydrogen-bonding network between the PΦB chromophore
and surrounding residues in the DrCBD protein pocket. Related statistics
presence/absence of the pyrrole water, (b) two distinct interac- are given in Tables 5 and 6.
tion distances between PΦB and HIS260 and (c) the out of plane
movement of the PΦB-ring A.
3.4. Hydrogen Network between the Chromophore and or during the MD simulation shorter that 3.4 Å31) are illustrated
Protein Pocket. To find out which residues in the vicinity the in Figure 10. The corresponding average donor-acceptor
chromophore play an important role in terms of hydrogen distances, evaluated over the 45 ns of the trajectory, are listed
bonding, average distances between hydrogen bond donor and in Table 5 together with the experimental values extracted from
acceptor between the chromophore and its surrounding amino the crystallographic structure for comparison.
acids were estimated. The most important hydrogen bond donor The agreement between measured distances from the X-ray
and acceptor pairs involving the chromophore and the protein and the average values obtained from the MD simulation is in
(H-bond with donor-acceptor distance in the X-ray structure general good, in particular for interactions 1, 2 and 3. For these
956 J. Phys. Chem. B, Vol. 113, No. 4, 2009 Kaminski et al.

TABLE 6: Statistics of H-Bond Events between the

Chromophore and Surrounding Residues with Occupancies
Larger Than 20%a
atom 1 atom 2 average occupancy
(protein) (PΦB) lifetime (ps) events (%)
ASP 207 O H_D 11.4 1436 36
ASP 207 O H_A 8.8 1016 21
ARG 254 HH11 O2D 60.8 541 73
ARG 254 HH11 O1D 90.9 169 34
ARG 254_HH21 O2D 32.9 930 68
ARG 254_HH21 O1D 26.4 1011 59
SER 257 HN O1D 146.3 201 65
HSE 260 HE2 O1A 405.6 90 81
SER 272 HG1 O2A 24.2 1028 55
SER 274 HG1 O1A 512.1 60 68
a
Statistical parameters were evaluated over 45 ns MD simulation.

TABLE 7: Statistics of H-Bond Events (with Occupancies

Larger Than 20%) between Important Water Molecules and
Residues in the Vicinity of the PΦB Chromophorea
average occupancy
water atom 2 lifetime (ps) events (%)
W1 O VAL 252 O 138.8 292 90
W1 O LEU 223 HN 43.0 823 79
W2 O ARG 254 HH12 13.6 1301 39
W2 H ARG 254 O 28.9 127 79
W2 H THR 256 OG1 24.6 1538 84
Figure 11. Stable water network in the chromophore binding pocket
W3 O TYR 216 HH 386.3 93 80
of Drph1. The structure refers to an average over the last 15 ns of the
simulation. Dotted lines indicate acceptor-hydrogen distances less than W3 H PΦB O2D 261.4 117 67
or equal than 2.4 Å. Related statistics are given in Tables 7 and 8. W5 O THR 224 HG1 1302.2 23 67
W5 H PΦB O2A 49.2 569 62
TABLE 5: Distances (Å) between Hydrogen Bond Donors W6 H PΦB O1A 22.7 1499 76
and Acceptors Participating in the Interactions Illustrated in W6 H PΦB CGA 9.5 2350 50
Figure 10a W7 H PΦB O1A 69.6 496 77
W7 H SER 272 OG 22.2 1273 63
H-bond X-ray MD ∆R W8 H PΦB O2A 18.6 1228 51
1 2.85 2.96 (0.42) 0.11 W8 O PΦB H_B 225.9 150 75
2 2.80 3.00 (0.33) 0.20 a
Statistical parameters were evaluated over 45 ns MD simulation.
3 2.99 2.89 (0.21) -0.10
4 2.67 4.21 (0.71) 1.54
5 2.64 3.31 (0.89) 0.67 side chain at ring C and the neighboring SER272, SER274 and
6 2.68 3.29 (0.51) 0.48 HIS260 residues (interactions 5, 6 and 7, respectively), sug-
7 2.80 3.20 (0.80) 0.40
8 4.99 4.49 (0.82) -0.50
gesting that this propionic side chain is looser than the one
9 2.96 3.44 (0.50) 0.48 attached on ring B, and probably less efficient for signal
10 2.77 3.28 (0.33) 0.51 transduction. Furthermore, these three interactions become
a
weaker during the simulation as indicated by the slight increase
Average distances and fluctuations (in parentheses) are
of the corresponding donor acceptor distances compared to the
calculated over the 45 ns of the simulation and compared to the
crystallographic structure. X-ray values. Large fluctuations are predicted for the interactions
7 and 8 involving ring A in PΦB with HIS260 which may be
related with the structural flexibility in this region and the
three interactions fluctuations of less than 0.40 Å are predicted, movement of the pyrrole water as previously discussed in section
indicating that the corresponding H-bonds remain stable during 3.3.
the simulation. These H-bonds formed between the chromophore The most pronounced discrepancy between the MD result
and ARG254 and SER257 are very important because they are and the X-ray H-bond donor-acceptor distance (∆R ) 1.54
responsible for the rigidity of the propionate side chain at ring Å) is found for interaction 4 defined between the TYR216 and
B. Furthermore, the two H-bond interactions with the Arginine the propionic side chain on ring B. The large average distance
residue are conserved in other phytochrome species, such as in of 4.21 Å indicates that the H-bond is broken during the MD
Cph129 and in RpBpphP3,30 and its rigidity may be of relevance simulation. In fact, a closer look at the trajectory shows that
for the signal transduction mechanism between chromophore for certain time spans a water molecule (W3 in Figure 11) places
and protein taking place during the photoinduced reaction cycle. itself between the TYR216 and the carboxylic group of the
The average H-bond donor-acceptor distances for the propionic side chain at ring B.
interactions 5-10 are slightly larger than the experimental ones Additional statistical information concerning the formation
(∆ ∼ 0.5 Å). In addition, most of these interactions (5-9) are of H-bonds in the chromophore binding pocket and their
associated with relatively high standard deviations (0.5-0.8 Å), lifetimes is summarized in Table 6. The statistical analysis was
reflecting a significant mobility of the residue side chains performed over the 45 ns MD simulation with a time resolution
involved in the corresponding interaction. This is in particular of 5 ps. The criteria for defining a hydrogen bond was that the
the case for the three H-bond interactions involving the propionic distance between hydrogen and the acceptor should be less or
MD Simulations of the Chromophore Binding Site of DrCBD
TABLE 8: Statistics of H-Bond Events between Important
Water Molecules Surrounding the Chromophore
water water average lifetime (ps) events occupancy (%)
W1 W2 39.1 905 79
W2 W4 6.9 2077 32
W3 W4 93.1 291 60
W4 W5 7.6 1816 31
W5 W6 33.0 738 54
W7 W8 16.8 1626 61
equal than a 2.4 Å.31 The stability of H-bonds formed between
the protein and the chromophore can be efficiently described
in terms of H-bond occupancy as the product between average
lifetime and number of events divided by the temporal length
of the MD simulation. The results presented in Table 6 show
that stable H-bonds are those formed between the ARG254 and
SER274 residues and the oxygen atoms at the propionic side
chain bound to ring B, with occupancies higher than 65%. This
salt bridge between the ARG254 and PΦB seem to be
responsible for rigidity of chromophore’s B ring. In a similar
way, the propionic side chain attached to ring C forms a stable
H-bond with the HIS260 and with the SER274, characterized
by long average lifetime of 405 and 512 ps, respectively, and
low number of events. The H-bonds formed between the
ASP207 and the NH groups of rings B and C, on the other
hand, do not seem to be very stable. They are constantly broken
and formed during the simulation, as indicated by the large
number of events, but each time they are formed they only exist
for a very short time (lifetime ∼ 10 ps). Thus the predicted
occupancies are fairly low (36% and 21%).
3.5. Water Network in the Chromophore Binding Pocket.
To investigate the formation of a water network in the
chromophore binding pocket, we selected eight water molecules
which remained during the production run within a 5 Å distance
from the chromophore. An illustration of the most stable water
network we identified is given in Figure 11, showing the
geometric average over the last 15 ns of the MD simulation.
Among these water molecules, only W3 is already observed in
the X-ray structure; W5, W7 and W8 are solvent waters that at
an early stage of the simulation occupy the position of a
crystallographic water, and the remaining water molecules are
solvent water that find a stable position in the vicinity of the
chromophore. As described in the previous section, the dynami-
cal behavior of this group of water molecules was analyzed by
performing a statistical analysis of the H-bonding events
involving the selected waters and calculating the so-called
H-bond occupancy. The results of the statistical analysis are
listed in Tables 7 and 8.
Very stable interactions are the H-bonds formed between W2
and the ARG 254 and THR256. These H-bonds are character-
ized by a large number of events (1127 and 1538) with average
lifetimes of ca. 25 ps and, consequently, large occupation factors
(79% and 84%). Similar results are found for the H-bond
interactions formed between W6 and the PΦB-propionic side
chain on ring C and between W7 and the SER272 residue. An
also very important interaction is that between THR224 and
W5. This water forms only few H-Bonds with the threonine,
but each of them is stable for 1.3 ns.
Furthermore, water W8 seems to be an important water bridge
connecting the chromophore’s ring D with the propionic side
chain attached to ring C. The H-bonds involving W8 and the
PΦB chromophore are very stable as indicated by the high
occupancy values (75% for the W8-PΦB(H_B) hydrogen bond
and 51% for the W8-PΦB(O2A) hydrogen bond). This water
J. Phys. Chem. B, Vol. 113, No. 4, 2009 957
bridge reduces the mobility of ring D in the Pr state. In addition,
W3, W5, W6 and W7 establish relatively stable H-bonds with
the two PΦB propionate side chains. The corresponding
hydrogen bond occupancies are larger than 50%. Although water
W4 is not H-bonded neither to the chromophore nor to the
apoprotein, its presence seems to be of fundamental importance
for the stability of the entire water network. This water is located
between the propionate side chains and interacts with W3, W2
and W5 with hydrogen bond occupancies larger than 30%. These
H-bonds are constantly formed and broken during the entire
production run; however, the relative positions of these water
molecules do not significantly change. The most stable
water-water interaction is that predicted between W1 and W2
with 79% H-bond occupancy. As shown in Figure 11, these
two water molecules do not directly interact with the chromophore.
Noticeable is the dynamical behavior of the crystallographic
pyrrole water. This water is located in the center of the
chromophore cavity forming H-bonds with the NH groups of
rings A, B and C. Our molecular dynamics simulations show
that these H-bonds are not strong enough to trap the pyrrole
water in the cavity for a long time. In particular, we observe
that the pyrrole water moves away from its initial position, and
after a while the empty space is filled up with a solvent water.
This process is repeated several times during the 45 ns
production run. As mentioned in the previous section, the
movement of the pyrrole water out of the chromophore cavity
significantly alters the chromophore structure.
4. Conclusions
In the present work, we could successfully extend the
CHARMM force field by developing parameters for phytocho-
mobilin (PΦB), a member of the biologically important family
of linear tetrapyrroles. The set of parameters reproduces in an
accurate manner the structural properties, rotational barriers and
intermolecular interactions of the chromophore as compared to
ab initio target data.
In addition, the quality of the new force field parameters was
tested by performing molecular dynamics simulations of the
entire bacteriophytochrome DrCDB. As a result, both structural
(average structure) and dynamic properties (root-mean-square-
fluctuations) of the chromophore are in satisfactory agreement
with the available experimental data. In contrast to the X-ray
structure where the chromophore’s rings A, B and C lie in a
coplanar arrangement, the MD simulations favors a twisted
conformation of the A-B methine bridge.
Furthermore, the MD simulations reveal the existence of
structural heterogeneity of PΦB chromophore in DrCBD. We
could identify two conformational states of the chromophore
characterized by distinct torsional angles around the C-C single
bond at the A-B methine bridge: 13.8° for state 1 and 18.5°
for state 2. The formation of these two states is associated with
the formation of hydrogen bonds between the residue HIS260
and the PΦB cofactor, as well as with the presence/ absence or
the pyrrole water in the chromophore cavity. The existence of
conformational heterogeneity involving ring A is in qualitative
agreement with NMR and RR spectroscopic data.
A statistical analysis of H-bonding events was performed to
study the dynamical behavior of H-bonds in the chromophore
binding pocket. Among the ten H-bond donor and acceptor pairs
considered in our analysis, those formed between the PΦB
chromophore and HIS260, ARG254 and SER274 residues are
in particular very stable. These H-bonds are responsible for the
rigidity of the propionic side chains at rings B and C and may
therefore of relevance for understanding the signal transduction
958 J. Phys. Chem. B, Vol. 113, No. 4, 2009
mechanism between chromophore and protein at an intermediate
stage of the photocycle.
Finally, the MD simulations allowed us to identify a stable
water network comprising at least eight water molecules in the
close vicinity of the chromophore. The presence of this water
network has a strong influence on the chromophore’s structural
properties. In particular, the propionic chains and ring D gain
rigidity from this network.
Additional Information
The parameter and topology files for PΦB are available from
the authors upon request.
Acknowledgment. We acknowledge the help from the
Konrad-Zuse-Zentrum in Berlin for supplying us with compu-
tational resources and we would like to thank Dr. Bernd Kallies
for technical support, as well as Prof. Peter Hildebrandt for
helpful discussions. The financial support by the Deutsche
Forschungsgemeinschaft, SfB498 to M.A.M and G.D. and the
financial support by the Investitionsbank Berlin and the Excel-
lence Cluster “Unifying concepts in catalysis” to S.K. and
M.A.M. is gratefully acknowledged.
Abbreviations
RR: resonance Raman
DrCB: Deinococcus radiodurans chromophore binding domain
PΦB: Phytochromobilin
QM: quantum mechanical
MM: molecular mechanical
MD: molecular dynamics
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