title: Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.
abstract: The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding.
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, 462023, India.
authors.forename: Mintu
authors.initials: M
authors.lastname: Chandra
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, 462023, India. Electronic address: sunando@iiserb.ac.in.
authors.forename: Sunando
authors.initials: S
authors.lastname: Datta
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides; D004748:Entamoeba histolytica / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D009711:Nucleotides; D020691:rab GTP-Binding Proteins; D003545:Cysteine
keywords: Cysteine; Inter-molecular disulfide bond; Intra-molecular disulfide bond; Oligomerization; Redox regulation