Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

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.

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