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T. Miyakawa et al.
are enhanced. In non-cancerous cell, GTP in Hras-GTP complex is hydrolyzed to
GDP in appropriate time. When inactive Hras-GDP complex is made, the propagating of the signals of cell division and cell differentiation is stopped [1].
To study the hydrolysis of GTP in Hras-GTP complex, we investigate the structure of Hras-GTP. In about 30 % of human cancer cells, at least one of Ras family,
which consists of Hras, Kras and Nras, is mutated [2]. The mutated Hras protein
has different structure from wild type Hras. This change of structure suppresses the
hydrolysis of GTP in Hras-GTP complex. As a result, the mutated Hras-GTP sends
continuously signals of cell division and cell differentiation [3]. The structure of
Hras-GTP complex is important for the hydrolysis of Hras-GTP. The suppression of
the hydrolysis of GTP is important to generate the tumor in the cell with the mutated
Hras-GTP complex. Investigating the structure of Hras-GTP complex is important
in order to prevent mutated Hras-GTP complex generating the tumor.
In 1990, the structures of Hras-GTP complex [4] and Hras-GDP complex
[5] were investigated by X-ray crystallography analyses. These analyses showed
that the structures of Hras-GTP complex and Hras-GDP complex are different in
switch I, which consists of 30–38 residues, and in switch II, which consists of 60–
72 residues.
In order to understand the mechanism of the hydrolysis of GTP in Hras-GTP
complex, we consider the role of water molecules in GTP hydrolysis by the analysis
of the position and direction of water molecules. The reaction mechanism of GTP
hydrolysis is classified mainly in two types. One is that the P–O bond is cleaved
after the attack of OH − . The other is that the P–O bond is cleaved and the water
molecule attacks the intermediate of hydrolysis. In order to know which mechanism
is preferred in Hras complex, we compare the positions of water molecules around
GTP and GDP. If some differences are found, the former mechanism is preferred.
While some researches suggest associative transition states [4, 6–12], other researches suggest mostly dissociative transition states [13–24]. We focused on the
orientation of water instead of OH − because most of these researches suggest that
GTPase catalyzes GTP hydrolysis reactions by neutralizing the negative charge development at β- or γ -phosphate of GTP and by correct positioning of the nucleophilic water molecule via a conserved Gln residue in the switch II region. Because
oxygen atoms in γ -phosphate of GTP are not protonated in Hras-GTP complex,
a water molecule attacks γ -phosphate in either case of dissociative mechanism or
associative mechanism.
In fact, the MD simulations of these complexes in water solvent have been performed in the former studies by several scientists [25–29]. In those studies, some
calculated the potential parameters with a small basis set [25, 26]. Others did not
write the used parameters in detail in their paper [27, 28]. Still others used the potential parameters of guanosine nucleotide calculated in water, not in Hras protein
complex [29].
Using the parameters in AMBER03 [30] and the parameters calculated by us
[31], we performed MD simulations of Hras-GTP and Hras-GDP complexes in water solvent. From the trajectories of MD simulations, we analyzed the position and
direction of water molecules.
T. Miyakawa et al.
are enhanced. In non-cancerous cell, GTP in Hras-GTP complex is hydrolyzed to
GDP in appropriate time. When inactive Hras-GDP complex is made, the propagating of the signals of cell division and cell differentiation is stopped [1].
To study the hydrolysis of GTP in Hras-GTP complex, we investigate the structure of Hras-GTP. In about 30 % of human cancer cells, at least one of Ras family,
which consists of Hras, Kras and Nras, is mutated [2]. The mutated Hras protein
has different structure from wild type Hras. This change of structure suppresses the
hydrolysis of GTP in Hras-GTP complex. As a result, the mutated Hras-GTP sends
continuously signals of cell division and cell differentiation [3]. The structure of
Hras-GTP complex is important for the hydrolysis of Hras-GTP. The suppression of
the hydrolysis of GTP is important to generate the tumor in the cell with the mutated
Hras-GTP complex. Investigating the structure of Hras-GTP complex is important
in order to prevent mutated Hras-GTP complex generating the tumor.
In 1990, the structures of Hras-GTP complex [4] and Hras-GDP complex
[5] were investigated by X-ray crystallography analyses. These analyses showed
that the structures of Hras-GTP complex and Hras-GDP complex are different in
switch I, which consists of 30–38 residues, and in switch II, which consists of 60–
72 residues.
In order to understand the mechanism of the hydrolysis of GTP in Hras-GTP
complex, we consider the role of water molecules in GTP hydrolysis by the analysis
of the position and direction of water molecules. The reaction mechanism of GTP
hydrolysis is classified mainly in two types. One is that the P–O bond is cleaved
after the attack of OH − . The other is that the P–O bond is cleaved and the water
molecule attacks the intermediate of hydrolysis. In order to know which mechanism
is preferred in Hras complex, we compare the positions of water molecules around
GTP and GDP. If some differences are found, the former mechanism is preferred.
While some researches suggest associative transition states [4, 6–12], other researches suggest mostly dissociative transition states [13–24]. We focused on the
orientation of water instead of OH − because most of these researches suggest that
GTPase catalyzes GTP hydrolysis reactions by neutralizing the negative charge development at β- or γ -phosphate of GTP and by correct positioning of the nucleophilic water molecule via a conserved Gln residue in the switch II region. Because
oxygen atoms in γ -phosphate of GTP are not protonated in Hras-GTP complex,
a water molecule attacks γ -phosphate in either case of dissociative mechanism or
associative mechanism.
In fact, the MD simulations of these complexes in water solvent have been performed in the former studies by several scientists [25–29]. In those studies, some
calculated the potential parameters with a small basis set [25, 26]. Others did not
write the used parameters in detail in their paper [27, 28]. Still others used the potential parameters of guanosine nucleotide calculated in water, not in Hras protein
complex [29].
Using the parameters in AMBER03 [30] and the parameters calculated by us
[31], we performed MD simulations of Hras-GTP and Hras-GDP complexes in water solvent. From the trajectories of MD simulations, we analyzed the position and
direction of water molecules.