FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

Abstract

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

FhCaBP1 (FH22): A Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains

“…2a). This is similar to the predicted structures of the other FhCaBP and SmTAL family members (Banford, et al, 2013, Orr, et al, 2012. In common with the other family members, the region linking the two domains is expected to be flexible, allowing the two domains to alter their relative orientation.…”

“…Thus, like other CaBP family members, FhCaBP1 can interact with this ion in addition to calcium. FhCaBP3 and FhCaBP4 both bind other ions in addition to calcium and manganese; however, like FhCaBP1, FhCaBP2 only binds these two ions (Banford, et al, 2013, Orr, et al, 2012). FhCaBP1's ability to bind calcium ions was confirmed by intrinsic fluorescence measurements and differential scanning fluorimetry.…”

“…We present the biochemical characterisation of FH22 (including predicted three-dimensional structure, ion binding, oligomerisation properties and interactions with calmodulin antagonists), building on previously reported work on this protein and complementing our own work on other family members (Ruiz de Eguino, et al, 1999) (Banford, et al, 2013, Nguyen, et al, 2016, Orr, et al, 2012. We report data on the protein's ion and drug binding properties, predicted structure and dimerization.…”

“…It forms a homodimer and calcium ion binding has no effect on dimer formation (Nguyen, et al, 2016, Thomas and. FhCaBP3 also interacts with calcium and manganese ions (Banford, et al, 2013). However in this case, calcium ion binding promotes the monomeric over the dimeric state (which is favoured in the absence of these ions).…”

“…Interestingly, in FhCaBP4 the situation is reversed: calcium ion binding promotes dimerization (Orr, et al, 2012). All three of these proteins interact with calmodulin antagonists and related drugs; however, the range of drugs which binds varies between the proteins, demonstrating that they can be distinguished pharmacologically (Banford, et al, 2013, Orr, et al, 2012.…”