Structural and thermodynamic characterization of metal binding in Vps29 from Entamoeba histolytica: implication in retromer function

• Published in: Molecular microbiology 2017-11, Vol.106 (4), p.562-581
• Main Authors: Srivastava, Vijay Kumar, Yadav, Rupali, Watanabe, Natsuki, Tomar, Priya, Mukherjee, Madhumita, Gourinath, Samudrala, Nakada‐Tsukui, Kumiko, Nozaki, Tomoyoshi, Datta, Sunando
• Format: Article
• Language: English
• Subjects: Alanine; Amino Acid Motifs; Amino Acid Sequence; Binding sites; Bound water; Calorimetry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cellular structure; Crystal defects; Crystal structure; Crystallography; Crystallography, X-Ray; Cysteine proteinase; Entamoeba histolytica; Entamoeba histolytica - genetics; Entamoeba histolytica - metabolism; Homology; Localization; Metallography; Metals; Models, Molecular; Mutants; Mutation; Protein Conformation; Protozoa; Secretion; Structural analysis; Structural integrity; Thermodynamics; Titration; Titration calorimetry; Trophozoites; Vesicular Transport Proteins - chemistry; Vesicular Transport Proteins - metabolism; Water chemistry; X-ray crystallography; Zinc
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13836

Summary Vps29 is the smallest subunit of retromer complex with metallo‐phosphatase fold. Although the role of metal in Vps29 is in quest, its metal binding mutants has been reported to affect the localization of the retromer complex in human cells. In this study, we report the structural and thermodynamic consequences of these mutations in Vps29 from the protozoan parasite, Entamoeba histolytica (EhVps29). EhVps29 is a zinc binding protein as revealed by X‐ray crystallography and isothermal titration calorimetry. The metal binding pocket of EhVps29 exhibits marked differences in its 3‐dimensional architecture and metal coordination in comparison to its human homologs and other metallo‐phosphatases. Alanine substitutions of the metal‐coordinating residues showed significant alteration in the binding affinity of EhVps29 for zinc. We also determined the crystal structures of metal binding defective mutants (D62A and D62A/H86A) of EhVps29. Based on our results, we propose that the metal atoms or the bound water molecules in the metal binding site are important for maintaining the structural integrity of the protein. Further cellular studies in the amoebic trophozoites showed that the overexpression of wild type EhVps29 leads to reduction in intracellular cysteine protease activity suggesting its crucial role in secretion of the proteases. EhVps29, a part of retromer complex localizes to the phagosomes in Entamoeba histolytica. It preferentially binds to zinc ion. Mutations of all the metal‐coordinating residues resulted in decrease in affinity for the zinc ion. The crystal structure of wild type and mutant of EhVps29 reveals that the metal positions are highly conserved and can be occupied with water. Moreover, the metal coordination in EhVps29 is distinct from the typical metallo‐phosphatases.