Structural and functional importance of a non-catalytic domain of FKBP35 from Plasmodium knowlesi

Jovi Silvester, (2019) Structural and functional importance of a non-catalytic domain of FKBP35 from Plasmodium knowlesi. Masters thesis, Universiti Malaysia Sabah.

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Abstract

A 35 kDa FK506-binding protein (FKBP35) from Plasmodium knowlesi (PkFKBP35) is considered as a viable target for development of antimalarial drugs without resistant effects. This protein is a member of peptidyl prolyl cis-trans isomerase (PPIase) with the ability to catalyze isomerization of cis-prolyl bond during protein folding. Sequence alignment of PkFKBP35 with other FKBP35 from P. falciparum and P. vivax revealed that PkFKBP35 consists of two domains which are the FK506-binding domain (FKBD) and the tetratricopeptide repeat domain (TPRD). FKBD acted as a catalytic domain, while TPRD serves as a non-catalytic domain. Development of new antimalarial drugs is so far focused only on the catalytic domain, while limited studies in the non-catalytic domain. Structurally, non-catalytic domain in other FKBPs was reported to be important for oligomerization of the proteins. There were also some cases that the oligomerization is associated with correct folding of the protein. Nevertheless, whether TPRD, as a non-catalytic domain of PkFKBP35, also structurally play important role for folding and dimerization remain to be investigated. Functionally, as the non-catalytic domain folds into TPR motif, thus this domain was thought to facilitate interaction between FKBP35 and other (partners) proteins. Since TPR motif in other proteins was known to interact to heat shock protein90 (Hsp90), it was also speculated that TPRD of FKBP35 might facilitate interaction between Plasmodium Hsp90, particularly to its C-terminal pentapeptide (MEEVD) and involved in folding machinery of the parasite cells. Interestingly, TPRD of FKBP35 segment contains a calcium-modulated proteins (calmodulin) binding motif (CBM) at its C-terminal. The presence of this motif promotes a speculation that TPRD might also interact with calmodulin and involved in calcium signaling pathway of the parasites. However, no study has been done to confirm these speculations. This study aims to determine the structural and functional roles of the non-catalytic domain (TPRD with its CBM) of PkFKBP35. Structural importance of non-catalytic domain was confirmed through solubility, folding and oligomerization assay. In addition, flexibility analysis revealed and 2D structural analysis of PkFKBP35 using transmission electron microscope revealed that PkFKBP35 was found to be a very dynamic protein with three conformations: circular, hook, elongated. This flexibility is believed regulated by catalytic domain. Further, binding analysis using pull down assay revealed the first evidences of interaction between PkFKBP35 and calmodulin (CaM). The binding was only observed in the presence of calcium ions which suggest that the interaction required an active state of CaM. Further analysis using surface plasmon resonance revealed that full length PkFKBP35 and PkTPRD+ bind to CaM with similar dissociation constant (KD values). This suggested that TPRD segment with its CBM is really essential for binding to CaM. In addition to the interaction to CBM, PkFKBP35 was also shown to be able to interact to MEEVD of Hsp90. This interaction was also found to be regulated by TPRD. Further, molecular docking analysis revealed that the binding sites of CaM are shared between TPRD and CBM. Altogether, the study demonstrated that non-catalytic domain has important role in protein-protein interaction function of PkFKBP35, mainly in facilitating the interaction to HSP90 or calmodulin. In addition, non-catalytic domain of PkFKBP35 is important for proper folding of this protein, yet, apparently, no involvement in structural flexibility of this protein.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Antimalarial drugs , protein , Plasmodium knowlesi
Subjects: Q Science > QD Chemistry
Depositing User: Noraini
Date Deposited: 11 Mar 2020 03:24
Last Modified: 11 Mar 2020 03:24
URI: http://eprints.ums.edu.my/id/eprint/25123

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