Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolyticaReportar como inadecuado

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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 and 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.

Autor: Shadab Anwar, Manas Ranjan Dikhit, Krishn Pratap Singh, Rajiv Kumar Kar, Amir Zaidi, Ganesh Chandra Sahoo, Awadh Kishore Roy, Tom



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