PNNL-13501 - Pacific Northwest National Laboratory

in the Environmental Molecular Sciences Laboratory. In
addition to the resources at MSCF, the DOE supercomputers
at the National Energy Research Scientific
Computing Center, Lawrence Berkeley National
Laboratory were also used to perform parts of the
computations.
Results and Accomplishments
Molecular dynamics simulations for p21Ras alone and in
complex with p120GAP were successfully completed
during the last year. In the p21Ras complexed with
p120GAP, only the Ras binding domain of GAP was
included in the simulations because only that part of the
protein is resolved in the x-ray experiments. The
molecular dynamics simulation systems were carefully set
up by first studying the protonation states (pKa) of the
protein residues and of the bound GTP. Our pKa
calculations showed that the protonation state of GTP
bound to p21Ras alone and bound to p21Ras:p120GAP
complex may be different. It was computed that the
terminal γ-phosphate group of GTP would be protonated
when p21Ras is alone, and it is unprotonated when
p21Ras forms a complex with p120GAP. Since it would
change the initial proton transfer to the GTP step of the
reaction, this difference in the protonation states implies
that the reaction mechanism of GTP to GDP hydrolysis
may be different for the p21Ras alone and
p21Ras:p120GAP systems.
Gln61 is believed to play an important role in the
structural stabilization of the catalytic site. In the
molecular dynamics simulation of p21Ras alone, Gln61
stays fully solvent exposed as in the experimental x-ray
structures. Gln61 is far from GTP, and therefore, most
likely does not get involved in the hydrolysis reaction
directly. It, however, contributes to the structural stability
of the local structure around the GTP binding pocket. In
contrast to the p21Ras alone system, Gln61 is close to the
GTP in the p21Ras:p120GAP complex and interacts with
GTP through a bridging water. It was observed in the
molecular dynamics simulations that Gln61 might be
occupying a position different than what was reported in
the x-ray structure. In its observed configuration, it
interacts with the GTP less specifically and contributes to
the hydrolysis reaction by helping to align the bridging
water in the proper orientation for the nucleophilic attack.
The x-ray crystal structure of the p21Ras:p120GAP
complex contains a water between Gln61 and GTP. This
water (we named it nucleophilic water) is believed to be
142 FY 2000 Laboratory Directed Research and Development Annual Report
the precursor of the nucleophilic hydroxyl that attacks the
γ-phosphate of GTP during the hydrolysis. In the crystal
structure, the nucleophilic water interacts with the
sidechain carbonyl group of Gln61. During our
simulation, there was a structural rearrangement of the
GTP binding pocket of the protein complex. During the
rearrangement, the nucleophilic water lost its interaction
with the carbonyl group of Gln61 and started to interact
with the NH2 sidechain group of the same residue. The
structure after the rearrangement supports the earlier
studies that Gln61 is probably not the general base in the
hydrolysis reaction.
The arginine residue (arginine finger) of p120GAP
pointing toward GTP may increase the GTPase activity of
Ras by stabilizing the transition state through favorable
electrostatic interactions. This hypothesis successfully
predicts that the GTPase activity of p21Ras increases
when it binds p120GAP and that the activity of Gα (with
its existing arginine finger) is higher than that of p21Ras.
It, however, cannot explain why the GTPase activity of
p21Ras:p120GAP is several orders of magnitude higher
than that of Gα even though both members of the systems
contain the arginine finger. In our p21Ras:p120GAP
molecular dynamics simulations, we have observed a
dominant structure of the GTP binding pocket. This
dominant configuration is quite different from the x-ray
crystal structure of the same complex. In this
configuration, the nucleophilic water moves into a
position such that its motion, orientation, and structural
stability is controlled by its interactions with the backbone
carbonyl of the arginine finger (Arg789 of p120GAP) and
with the NH2 sidechain group of Gln61. In the
corresponding structure of Gα, the water molecule does
not interact with the backbone of the arginine finger
residue. Based on our observations, we have postulated
that, in addition to its transition state stabilization role as
in the Gα system, the arginine finger of p120GAP also
controls the orientation of the nucleophilic water while
initiating the hydrolysis reaction. It is the single-handed
operation of orienting the attacking water and the
transition state stabilization by the same residue that
makes the GTPase activity of p21Ras:p120GAP complex
higher than that of the Gα system.
Summary and Conclusions
Protonation state and molecular simulation computations
of the p21Ras and the p21Ras:p120GAP complex were
successfully completed. We found that the protonation
state of the bound GTP may be different in these two