Metal binding affinity and structural properties of an isolated EF-loop in a scaffold protein

Abstract

To establish an approach to obtain the site-specific calcium binding affinity of EF-hand proteins, we have successfully designed a series of model proteins, each containing the EF-hand calcium-binding loop 3 of calmodulin, but with increasing numbers of Gly residues linking the loop to domain 1 of CD2. Structural analyses, using different spectroscopic methods, have shown that the host protein is able to retain its native structure after insertion of the 12-residue calcium-binding loop and retains a native thermal stability and thermal unfolding behavior. In addition, calcium binding to the engineered CD2 variants does not result in a significant change from native CD2 conformation. The CD2 variant with two Gly linkers has been shown to have the strongest metal binding affinity to Ca(II) and La(III). These experimental results are consistent with our molecular modeling studies, which suggest that this protein with the engineered EF-loop has a calmodulin-like calcium binding geometry and backbone conformation. The addition of two Gly linkers increases the flexibility of the inserted EF-loop 3 from calmodulin, which is essential for the proper binding of metal ions.

Identification of a Ca 2+ -Binding Domain in the Rubella Virus Nonstructural Protease

“…The predicted 12-residue Ca 2ϩ -binding loop was directly inserted between Gly53 and Ala54 by PCR using an established protocol (70). The mutant with double mutations D5A and D12A (numbered according to the loop position of the inserted Ca 2ϩ -binding motif) was produced using standard PCR methods.…”

“…The concentration of RUBCa was measured by its absorption at 280 nm with an extinction coefficient of 19,630 M Ϫ1 cm Ϫ1 , which was calculated according to previously described methods (22). The engineered proteins CD2.RUBCa and the mutant CD2.RUBCa.D5A/D12A were expressed and purified according to methods described previously (70). The molecular weights of CD2.RUBCa and the D5A/D12A mutant were also confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.…”

“…2A, CD2 is a cellular adhesion molecule composed of nine ␤-strands with an immunoglobulin-like fold, and the putative Ca 2ϩ -binding loop from the RUB NS protease was inserted between Gly53 and Ala54, which lie within the loop between strands CЉ and D of CD2; the grafted construct was termed CD2.RUBCa. Additionally, three glycines were placed on each side of the Ca 2ϩ -binding loop to serve as linkers to provide sufficient conformational freedom for the grafted Ca 2ϩ -binding loop and to minimize perturbation on the host protein while allowing the inserted loop to retain its capability of chelating Ca 2ϩ (31,(69)(70)(71). The spectroscopic silence of Ca 2ϩ makes it extremely challenging to investigate Ca 2ϩ -binding properties directly.…”

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor

“…The geometrically predicted locations with 2-4 negatively charged ligands, strong negative surface potential, and functionally necessary residues were more likely to bind Ca 2ϩ . The model structures of CD2 variants with grafted potential Ca 2ϩ -binding sites of the CaSR were generated by SWISSMODEL (33,34 52 and Gly 53 of CD2 in the plasmid pGEX-2T (denoted as CD2-CaSR1 and CD2-CaSR2, respectively) by PCR using an established protocol (63). Two mutants, CD2-CaSR1-E228A/E229A and CD2-CaSR2-E378A/E379A, were also made by site-directed mutagenesis.…”

Conserved properties of individual Ca 2+ -binding sites in calmodulin

“…Different EF-hand-containing proteins bind Ca 2+ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca 2+ with a different affinity at each site (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). It has therefore been suggested that CaM's four sites display different affinities and perhaps cooperativity (29,30).…”