these systems was of ca. 75,000 and 150,000 atoms, respectively for the monomer
and dimer.
Each of the two systems was subjected to a 4-stages refinement protocol using
the SANDER module of AMBER 10, in which the constraints on the enzyme were
gradually removed. In the first stage (10,000 steps), 50 kcal/mol/Å
2 harmonic forces
were used to restrain the positions of all atoms in the systems except those from the
water molecules. In the second stage (10,000 steps) these constraints were applied
only to the heavy atoms, and in the third stage (30,000 steps), they were limited to
the CA and N atom-type atoms (backbone alfa carbon atoms and nitrogen atoms).
This process ended with a full energy minimization (4th stage, maximum 80,000
steps) until the rms gradient was smaller than 0.02 kcal/mol/Å
2
.
MD simulations were carried out using the PMEMD module of AMBER 10, and
considering periodic boundary conditions to simulate a continuous system. The
SHAKE algorithm [64] was applied to fix all bond lengths involving a hydrogen
bond, permitting a 2 fs time step. The Particle-Mesh Ewald (PME) method [65] was
used to include the long-range interactions, and a non-bond interaction cut-off
radius of 10 Å was considered. Following a 40 ps equilibration procedure, 10 ns
MD simulations were carried out at 310 K for each of the 2 systems (a total of
20 ns), using Langevin temperature coupling and constant pressure (1 atm) with
isotropic molecule-based scaling. The MD trajectory was sampled every 2 ps. All of
the MD results were analyzed with the PTRAJ module of AMBER 10.
The ConSurf server [66] was used to obtain estimated conservation grades for
each amino acid position along the FAAH sequence. This server uses the initial 3-D
structure of a given protein or domain (in this case the chain A in the 1MT5 PDB
structure [3]) and carries out a search for close homologous sequences using the
PSI-Blast [67] heuristic algorithm, with default parameters, to collect homologous
sequences of a single polypeptide chain of known 3D-structure from the SWISSPROT database [68]. These sequences are then aligned against the sequence of the
initial protein with the MUSCLE algorithm [69]. A conservation score is calculated
along each amino acid position using an empirical Bayesian [70] approach. The
ConSurf-HSSP database may be accessed at http://consurf-hssp.tau.ac.il.
3 Results and Discussion
This section describes the broad analysis on the FAAH-anandamide complex of
both monomer and dimer complexes. First, a description of the stability of both
systems is shown through a Root Mean Square Displacement analysis (RMSD).
Root Mean Square Fluctuation (RMSF) values on both monomer and dimer
complexes are also presented, allowing an analysis on the flexibility patterns of
both systems. Finally, and in order to characterize the interaction of FAAH and the
anandamide substrate, SASA analysis, Radial Distribution Functions (RDFs) of the
water around the substrate, and hydrogen bond analysis on the active-site are
additionally conducted.
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S.F. Sousa et al.
and dimer.
Each of the two systems was subjected to a 4-stages refinement protocol using
the SANDER module of AMBER 10, in which the constraints on the enzyme were
gradually removed. In the first stage (10,000 steps), 50 kcal/mol/Å
2 harmonic forces
were used to restrain the positions of all atoms in the systems except those from the
water molecules. In the second stage (10,000 steps) these constraints were applied
only to the heavy atoms, and in the third stage (30,000 steps), they were limited to
the CA and N atom-type atoms (backbone alfa carbon atoms and nitrogen atoms).
This process ended with a full energy minimization (4th stage, maximum 80,000
steps) until the rms gradient was smaller than 0.02 kcal/mol/Å
2
.
MD simulations were carried out using the PMEMD module of AMBER 10, and
considering periodic boundary conditions to simulate a continuous system. The
SHAKE algorithm [64] was applied to fix all bond lengths involving a hydrogen
bond, permitting a 2 fs time step. The Particle-Mesh Ewald (PME) method [65] was
used to include the long-range interactions, and a non-bond interaction cut-off
radius of 10 Å was considered. Following a 40 ps equilibration procedure, 10 ns
MD simulations were carried out at 310 K for each of the 2 systems (a total of
20 ns), using Langevin temperature coupling and constant pressure (1 atm) with
isotropic molecule-based scaling. The MD trajectory was sampled every 2 ps. All of
the MD results were analyzed with the PTRAJ module of AMBER 10.
The ConSurf server [66] was used to obtain estimated conservation grades for
each amino acid position along the FAAH sequence. This server uses the initial 3-D
structure of a given protein or domain (in this case the chain A in the 1MT5 PDB
structure [3]) and carries out a search for close homologous sequences using the
PSI-Blast [67] heuristic algorithm, with default parameters, to collect homologous
sequences of a single polypeptide chain of known 3D-structure from the SWISSPROT database [68]. These sequences are then aligned against the sequence of the
initial protein with the MUSCLE algorithm [69]. A conservation score is calculated
along each amino acid position using an empirical Bayesian [70] approach. The
ConSurf-HSSP database may be accessed at http://consurf-hssp.tau.ac.il.
3 Results and Discussion
This section describes the broad analysis on the FAAH-anandamide complex of
both monomer and dimer complexes. First, a description of the stability of both
systems is shown through a Root Mean Square Displacement analysis (RMSD).
Root Mean Square Fluctuation (RMSF) values on both monomer and dimer
complexes are also presented, allowing an analysis on the flexibility patterns of
both systems. Finally, and in order to characterize the interaction of FAAH and the
anandamide substrate, SASA analysis, Radial Distribution Functions (RDFs) of the
water around the substrate, and hydrogen bond analysis on the active-site are
additionally conducted.
118
S.F. Sousa et al.