UC Riverside Undergraduate Research Journal

Computational Prediction of Association Free Energies for the C3d-CR2 Complex and Comparison to Experimental Data
Alexander S. Cheung
methionine of C3d which is an artifact added by the protein
expression system. One of the chains in the PDB file, chain
C, a CR2 molecule that is not in contact with C3d, was also
removed. This is because chain C is irrelevant in this study,
since it is known that CR2 behaves as a monomer in solution
[7]. What remains is a C3d molecule, consisting of 306 amino
acids, in contact with a CR2 molecule, consisting of 129
amino acids. We used the program SPDBV (Swiss Protein
Data Bank Viewer) [17], version 3.7, to renumber the amino
acids and atoms in the PDB file (displacing every atom by
-1, resulting in a total of 435 amino acids consisting of 3399
atoms) and subsequently separate the two components of the
complex to create one PDB file with C3d alone and one PDB
file with CR2 alone. After separation, amino acids and atoms
were renumbered for CR2 to begin at residue 307 and atom
2413. Thus three PDB files constitute the final output of
this step: one PDB file consisting of the C3d-CR2 complex,
one consisting of C3d, and one consisting of CR2. These
three PDB files are considered the “parent” PDB files. By
generating each of the component parent PDB files from the
complex parent PDB file in this way, it is ensured that the
atomic coordinates of each component of the complex in
each of their respective component files are identical to their
atomic coordinates in the complex file. This is crucial for
the accurate calculation of free energy differences. Finally,
we used the program WHATIF [18] to add the missing
C-terminal oxygen atom of C3d in the C3d-CR2 complex.
The third step (Fig. 2) was the construction of the 23
specific mutants. This is done using WHATIF, a home-made
python script [19] that calls WHATIF, the three parent PDB
files, and three input text files, each of which list the amino
acid substitutions to be made in one of the three parent PDB
files. Each of the 23 mutations had to be performed twice:
once on the complex PDB file and again on the individual
component file containing the mutation(s). Thus, the script
was run three times, each time using as inputs one of the
three parent PDB files and its corresponding input text file.
For the purpose of consistency, each of the parent PDB files
was also run through WHATIF manually without making any
mutations. The outputs of this step are 23 mutant complex
PDB files, 9 mutant C3d PDB files, 14 mutant CR2 PDB
files, and the three parent PDB files (49 PDB files total).
These 49 PDB files comprise 24 sets (parent and 23 mutants)
of 3 PDB files each (C3d, CR2, complex).
The fourth step (Fig. 2) was the removal of a
WHATIF-specific header added to each PDB file in the
last step, and the change of the nomenclature of C-terminal
oxygens from O’’, which is recognizable by WHATIF, to
OXT, which is recognizable by PDB2PQR (to be used in
the next step). These two tasks are accomplished using
home-made python scripts [19].
The fifth step (Fig. 2) involved the use of the
program, PDB2PQR [20] 1.2.1, to prepare the coordinate
PDB files for use with APBS (see below). Through the use
of a home-made python script, each of the 49 PDB files was
run through PDB2PQR. The outputs were 49 files in PQR
format, each containing three-dimensional atomic coordinate
data as well as charge and van der Waals radii assigned
according to the PARSE parameter file [20]. The default
options for debumping and hydrogen bond optimization
were left on. Debumping refers to local optimization to
eliminate unfavorable van der Waals clashes (overlap or
partial overlap of atomic radii). The hydrogen bond network
optimization algorithm assures that optimal hydrogen bonds
are present by 180 o -flipping the rings of histidine or of planar
amine groups of glutamines or asparagines. This option is
necessary because electron densities from X-ray diffraction
data do not discriminate between the 0 o - and 180 o -flip states
of these amino acid side chains.
The purpose of steps 1-5 described above is to
create the proper input files for use with the program
APBS (Adaptive Poisson-Boltzmann Solver) [22]. The
sixth step was calculation of electrostatic potentials using
Figure 3. Hypothetical thermodynamic cycle. Horizontal processes
represent association in vacuum (top) and in solution (bottom).
Vertical processes represent solvation of the components (left)
and of the complex (right). Electrostatic potential surfaces are
visualized at ±30 kT/e for association in vacuum (top) and at ±1
kT/e in solution (bottom).
16 UCR Un d e r g r a d u a t e Re s e a r c h Jo u r n a l