A R T I C L E S
Lund et al.
Table 1. Definition of the Complexes with Different Ligands Bound
The net negative charge of the protein was neutralized by added
Na+ ions. Explicit TIP3P waters were added as a truncated
octahedral water box with a 10 Å buffer.45 All operations were
processed using the AMBER Leap module, which led to models
containing ∼70 000 atoms, including ∼19 000 waters.46,47 These
models were further extended using the periodic boundary condition.
The AMBER force field 99,48 with the parameters for the peptide
backbone reoptimized, was utilized for all standard amino acid
residues while the general AMBER force field was used for
carbamate.49 The force fields for ADP, Pi, and Mn2+ were
previously developed for the Amber program.50,51 MD simulations
were conducted in an isothermal-isobaric (NPT) ensemble at 300
K and 1 atm. The SHAKE algorithm was used to constrain all bonds
involving hydrogens.52 A 10 Å cutoff was applied for nonbonding
interactions. The Particle Mesh Ewald method was employed to
treat long-range electrostatic interactions.53,54
substrate(s)
name
PDB code
site 1a
site 2b
Arg-306c
A
B
C
1bxr
1bxr
1c30
ADP
ADP + Pi
ADP + Pi
ATP
ATP
ADP
open
closed
open
a Site for carbamate synthesis. b Site for carbamoyl phosphate
synthesis. c When Arg-306 is hydrogen bonded to the phosphate site 1
the tunnel is closed and it is open when this residue is hydrogen bonded
to Glu-25, Glu-383, and Glu-604.
site of formation in the N-terminal domain to the site of
utilization in the C-terminal domain of CPS.
Materials and Methods
MD trajectories were obtained with these structures using the
AMBER suite of programs.46,47 Before the MD simulations, in
which the trajectories were collected every 0.5 ps, two steps of
minimization were conducted. During minimization, the systems
were first optimized for 1000 cycles while the backbone of the
Construction of Models for the Transport of Carbamate. The
large subunit from the X-ray crystal structure of CPS (PDB codes:
1c30 and 1bxr) was taken as the starting point for the simulations
of carbamate transfer in CPS.31,32 In one of these structures (PDB
code: 1c30) ADP and phosphate are bound in the active site for
carbamate formation whereas ADP alone is bound in the active
site for the synthesis of carbamoyl phosphate. In the other structure
(PDB code: 1bxr) AMPPNP, an ATP analogue, is bound to both
active sites in the large subunit of CPS. Two missing loops (residues
717-723 and 742-749) in the large subunit of CPS (PDB code:
1c30) were inserted using SWISS-MODEL.33-35 These two loops
are part of the binding site for ATP in the carbamoyl phosphate
synthesis domain. Without the γ-phosphate of the substrate, the
domain containing these two loops is apparently flexible. In the
large subunit, obtained from the structure represented by PDB
code 1bxr, the bound AMPPNP was substituted with ATP in the
active site for the phosphorylation of carbamate. Conversely, in
the active site for the phosphorylation of bicarbonate, AMPPNP
was substituted with ADP and phosphate, or ADP alone. Three
structural complexes were created for the simulations and these
are denoted as complexes A, B and C in Table 1.
The charge distributions on all atoms in carbamate were obtained
using the RESP-fit method36,37 based on B3LYP/cc-pVTZ
calculations38-41 with the solvent effect (ꢀ ) 4) taken into account
using the polarizable continuum model (PCM).42-44 Before charge
fitting, the geometry for carbamate was fully optimized in the gas
phase at the same theoretical level. The charges on the carbon,
hydrogen, oxygen, and nitrogen atoms in carbamate are +0.883517,
+0.325533, -0.824983, and -0.884618, respectively. Mn2+ and
all of the crystalline waters within the enzyme or on the protein
exterior were utilized for the simulations. The protonation states
of all histidine residues were adjusted based on local environments.
protein was frozen with a force constant of 500 kcal·mol-1 ·Å-2
.
The systems were further optimized for 2500 cycles without
constraints, followed by a 20-ps MD simulation which heats the
system from 0 to 300 K, with a force constant of 10 kcal ·mol-1 ·Å-2
added upon the protein backbone. A 200-ps simulation under 1
atm and 300 K was performed to reach equilibrium with the
constraints on the protein removed.
Free Energy Calculations. The reaction coordinate for the free-
energy simulations was defined as the distance from the carbon
atom (Ccbm) of carbamate to the ꢀ-phosphoryl group of ADP in
the active site for carbamate formation as shown in Figure 2. The
free-energy profiles or potentials of mean force (PMF) were
computed along the translocation trajectories using the umbrella
sampling technique with a biasing harmonic potential involving a
force constant of 40 kcal ·mol-1 ·Å-2 55-59 A total of 113 windows
.
were chosen for each of the three systems with a step size of 0.25
Å starting from 5 Å and ending at 33 Å. An equilibration simulation
for each window was performed and the corresponding PMF along
the reaction coordinate was computed every 500 ps using the
weighted histogram analysis method (WHAM)56-58 until conver-
gence was observed. The last four 500-ps trajectories, without
yielding significantly different PMF when used separately, were
combined as a 2-ns simulation for WHAM analysis. Because of
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3872 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010