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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
66
7-POS
Board 7
Insights into Molecular Switch: Crystal Structure Analysis of Wild Type and Fast
Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba
Histolytica
Mintu Chandra
1
, Vijay K. Srivastava
1
, Yumiko S. Nakano
2
, Tomoyoshi Nozaki
2
.Sunando
Datta
1
1
Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Indiana,
India,
2
National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Shinjuku-ku, Tokyo,
Japan.
Background: The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of
amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in
the survival and virulence of the protozoan and is regulated by various Rab GTPases.
EhRabX3, a catalytically inefficient amoebic Rab protein, is unique among the eukaryotic Ras
superfamily by virtue of its tandem domain organization. A detail structural analysis revealing
sub-atomic description of the protein is essential for understanding how the classical GTP/GDP
cycle is influenced by the domain organization in 3-dimensional space and why this atypical
GTPase is catalytically dead.
Methodology/Results: Here, using Se-Met SAD phasing, we report the crystal structures of GTP-
bound wild type EhRabX3 and its fast hydrolyzing mutant in GDP-bound form. Interestingly,
only the N-terminal G-domain showed nucleotide bound to its active site. The overall structure
showed that the central beta sheets of NTD and CTD are oriented at right angle to each other.
Compared to other Ras superfamily members, both G-domains displayed large deviation in
switch II, perhaps due to non-conservative substitution in this region. The bulky side chain of
Val71 occluded Lys73 from the nucleotide binding pocket, providing a rationale for the
diminished GTPase activity of EhRabX3. Replacement of Val71 and Lys73 by conventional
counterparts, Ala and Gln, respectively, resulted in large enhancement in its GTPase activity.
Moreover, the existence of an intra-molecular disulfide bond in EhRabX3 is found to be critical
for maintaining the structural integrity and function of this unique Rab protein.
Conclusions: Structure-guided functional investigation of cysteine mutants could provide
physiological implications of the disulfide bond and allow us to design potential inhibitors for
the better treatment of intestinal amoebiasis.