title: Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.
abstract: Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Shadab
authors.initials: S
authors.lastname: Anwar
authors.affiliation: Department of Biomedical Informatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Manas Ranjan
authors.initials: MR
authors.lastname: Dikhit
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Krishn Pratap
authors.initials: KP
authors.lastname: Singh
authors.affiliation: Department of Biomedical Informatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Rajiv Kumar
authors.initials: RK
authors.lastname: Kar
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Amir
authors.initials: A
authors.lastname: Zaidi
authors.affiliation: Department of Biomedical Informatics Centre, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Ganesh Chandra
authors.initials: GC
authors.lastname: Sahoo
authors.affiliation: Department of Botany, T. M. Bhagalpur University, Bhagalpur, India.
authors.forename: Awadh Kishore
authors.initials: AK
authors.lastname: Roy
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam-kuan, Patna, India.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins