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Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A
journal contribution
posted on 2017-10-12, 12:35 authored by Chaithanya Kotyada, Aditi Maulik, Anand Srivastava, Mahavir SinghRheB GTPase is a Ras-related molecular
switch, which regulates
the mTOR signaling pathway by cycling between the active [guanosine
triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)]
state. Impairment of GTPase activity because of mutations in several
small GTPases is known to be associated with several cancers. The
conventional GTPase mechanism such as in H-Ras requires a conserved
glutamine (Q64) in the switch-II region of RheB to align the catalytic
water molecule for efficient GTP hydrolysis. The conformation of this
conserved glutamine is different in RheB, resulting in an altered
conformation of the entire switch-II region. Studies on the atypical
switch-II conformation in RheB revealed a distinct, noncanonical mode
of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively
enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A
double mutant was shown to reduce the elevated GTPase activity. Here,
we have used all-atom molecular dynamics (MD) simulations for comprehensive
understanding of the conformational dynamics associated with the fast
(Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination
of starting models from PDB structures and in-silico generated mutant
structures, we discuss the observed conformational deviations in wild
type (WT) versus mutants. Our results show that a number of interactions
of RheB with phosphates of GTP as well as Mg2+ are destabilized
in Y35A mutant in the switch-I region. We report distinct water dynamics
at the active site of WT and mutants. Furthermore, principal component
analysis showed significant differences in the conformational space
sampled by the WT and mutants. Our observations provide improved understanding
of the noncanonical GTP hydrolysis mechanism adopted by RheB and its
modulation by Y35A and Y35A-D65A mutants.