Atypical Switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica Chaithanya Kotyada a , Mintu Chandra b , Aashutosh Tripathi c , Anil R. Narooka c , Sunando datta c, * , Akash Verma d a Biological Sciences, The University of Auckland, Auckland, 1010, New Zealand b Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Brisbane, 4072, Australia c Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, 462066, India d Maa Smriti Bhavan, Lal Kothi Compound, Hazaribagh, 825301, India article info Article history: Received 13 October 2018 Accepted 17 October 2018 Available online 27 October 2018 Keywords: Rab GTPase Intrinsic GTP hydrolysis Switch-I Switch-II abstract Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005 ]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015 ]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Muta- tions that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine. © 2018 Elsevier Inc. All rights reserved. 1. Introduction Ras superfamily GTPases are ubiquitous molecular switches involved in a variety of cellular processes including cell prolifera- tion, cytoskeletal dynamics and intracellular vesicle trafficking. They alternate between the GTP and GDP bound conformations in the cell where the GTP bound “ON” conformation is biologically active and turns on the cellular functions through interaction with specific effector(s). GTP hydrolysis turns “OFF” the GTPase switch by converting it to the inactive GDP bound conformation that has low affinity for the effector(s). The ability to bind and hydrolyze GTP is determined by primary amino acid sequence motifs which are highly conserved evolutionarily [4]. Small GTPases of Ras super- family can be recognized by five conserved fingerprint sequence motifs; the G1 box, also known as P-loop with purine nucleotide binding signature motif, GxxxxGKT/S; G2 box of switch-I region with a conserved Threonine; G3 box with DxxGQ motif, also known as switch-II; residues of the G4 box and G5 box having NKxD and SAK/L motifs respectively which primarily make associations with guanine nucleotide and therefore are the major determinants of guanine nucleotide specificity [5]. It has been suggested that the conserved Glutamine residue in the switch-II region is required to stabilize the hydrolytic water molecule for the SN 2 like ‘in-line’ nucleophilic attack on the g-phosphate of GTP [3,6e10]. Mutation of this residue increases the activation barrier for hydrolysis and thereby decreasing the GTP hydrolysis rate [11 , 12]. The enteric protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery, liver abscess and colitis in humans [13]. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. Entamoeba genome encodes nearly hundred Rab GTPases, perhaps reflecting a complex vesicular trafficking network [1]. Out of such a repertoire of Rabs present in genome, 22 of them showed * Corresponding author. E-mail address: sunando@iiserb.ac.in (S. datta). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.10.113 0006-291X/© 2018 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 506 (2018) 660e667