Functional manipulation of a calcium binding protein from E. histolytica guided by paramagnetic NMR.
J Biol Chem. 2013 Jun 19;
Authors: Rout AK, Patel S, Gupta S, Shukla M, Saraswathi D, Bhattacharya A, Chary KV
Abstract
EhCaBP1, one of the calcium binding proteins from E. histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both the domains are required for structural stability and full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses unique structure compared to other family members. This had been attributed to the absence of Phe-Phe interaction between highly conserved Phe residues at -4th position in the EF-hand III (F[-4]; Y81) and that at the 13th position in the EF-hand IV (F[+13]; F129) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at -4th position of EF III to Phe residue (Y81F), to bring in Phe-Phe interaction and to understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC) and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favoured by the presence of ?-? interaction between F81 and F129 along with several hydrophobic interactions of F81, which are not seen in the wild type protein. Further, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups while retaining its ability to binding G- and F-actin.
PMID: 23782698 [PubMed - as supplied by publisher]
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Functional manipulation of a calcium binding protein from E. histolytica guided by paramagnetic NMR.
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