Functional and structural analysis of the conserved EFhd2 protein

Abstract

EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2's calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2's thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2's thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins.

Fig. 1. His-EFhd2^WT and truncation mutants His-EFhd2^ΔNT and His-EFhd2^ΔCC bind calcium in vitro

A) Illustrated representation of EFhd2 domains under study, the N-terminus, the polyalanine domain, within the central region of the protein, the two EF-hand motifs and the coiled-coil domain at the C-terminus of the protein. The illustration is not at scale. B) Recombinant His-EFhd2^WT, His-EFhd2^ΔNT and His-EFhd2^ΔCC were purified and incubated with radioactive ⁴⁵CaCl₂ to determine their calcium binding activity. Nickel-column beads were incubated with un-induced bacterial extract as negative control (His 6X). The radioactivity remaining in the beads was measured on a scintillation counter in counts per minute (CPM). C) A gel representative of the proteins used in this assay is illustrated. Protein amount was used to normalize the level of calcium binding detected. Significance was determined by a student’s T-test (two-tailed, paired). Statistical significance of * p < 0.01 and ** p < 0.001 is indicated.

Fig. 2. EFhd2 EF-hand motifs are conserved throughout evolution

Sequence alignment of EFhd2 calcium binding motifs (1^st and 2^nd EF-hand motif) was performed using sequences from various species. The aspartates (D) in bold represent the first amino acid within each EF-hand motif loop (EF-loop) that is essential for the domain’s calcium-binding activity. These aspartate residues were changed to alanine (A) by introducing a point mutation in the cDNA sequence. The phenylalanine residue (F) in the first EF-hand represents a conserved amino acid affected by the identified SNP rs12131549. The SNP introduces a missense mutation that changes the phenylalanine to a leucine (L).

Fig. 3. Both EF-hand domains are required for EFhd2 calcium-binding affinity

A) Recombinant proteins HIS-EFhd2^WT, HIS-EFhd2^F89L, HIS-EFhd2^D105A and HIS-EFhd2^D141A were subjected to in vitro calcium binding assay using radioactive ⁴⁵CaCl₂. Nickel-column beads were incubated with un-induced bacterial extract as negative control (His 6X). The amount of radioactive calcium was measured with a scintillation counter in counts per minute (CPM). B) The amount of recombinant protein was used to normalize the level of calcium binding. Significance was determined by a student’s T-test (two-tailed, paired). Statistical significance of * p< 0.02 and **p<0.01 is indicated.

Fig. 4. His-EFhd2^WT, His-EFhd2^ΔNT and His-EFhd2^ΔCC secondary structure revealed by circular dichroism

A–D) Circular dichroism was used to determine changes upon calcium binding in the secondary structure of recombinant His-EFhd2^WT and truncation mutants at 25°C. The secondary structure of BSA (A), His-EFhd2^WT (B), His-EFhd2^ΔNT (C) and EFhd2^ΔCC (D) was analyzed in the far-UV region (200–260 nm) without calcium (black solid line) and with 1mM of CaCl₂ (dashed line). Bovine serum albumin was used as control for instrument precision and as a negative control for structural changes after addition of calcium.

Fig. 5. Thermal stability of EFhd2^WT

A–D) Thermal stability studies on His-EFhd2^WT were conducted using circular dichroism. Measurements of the protein in the far-UV region (200–260 nm presented) were taken every 10°C from 25°C to 75°C. Two representative temperature spectra at 25°C (solid line) and 75°C (dashed line) are shown. The thermal stability of BSA (used as positive denaturation control) was measured in absence (A) or presence (B) of 1mM CaCl_2. His-EFhd2^WT was analyzed in the absence (C) or presence (D) of 1mM CaCl₂ using the same temperature range. The representative spectra at 25°C (solid line) and 75°C (dashed line) are shown.

Fig. 6. The N-terminus and calcium binding are required for EFhd2’s thermal stability

Recombinant His-EFhd2^ΔNT and His-EFhd2^ΔCC secondary structures were analyzed by circular dichroism to determine the contribution of the N- and C-terminus on EFhd2 thermal stability. Measurements were taken every 10°C from 25°C to 75°C in the far-UV region (200–260 nm). Two representative spectra, 25°C (solid line) and 75°C (dashed line) are shown. The effect of temperature on the structure of His-EFhd2^ΔNT was determined in the absence (a) or presence (b) of 1mM CaCl₂. The same analyses were performed for His-EFhd2^ΔCC without calcium (c) or with (d) 1mM CaCl₂. Deconvolution of the CD spectra obtained for His-EFhd2^ΔNT and His-EFhd2^ΔCC spectra are presented in Tables 3 (without calcium) and 4 (plus calcium)

Fig. 7. Structural prediction of the EFhd2 protein

The mouse protein sequence of EFhd2 was analyzed using the protein structure prediction algorithms Phyre2. The predicted structures of EFhd2 wild-type (WT) protein, N- (ΔNT) and C-terminus (ΔCC) deletion mutants and EFhd2^F89L mutant are illustrated. The position of the conserved aspartates (D105 and D141), is indicated (arrows). Dashed arrows indicate that D105 and D141 are behind the plane. The distance (Å) between amino acids in the same EF-hand loop (D105-D116) and adjacent EF-hand motifs (D105-D141) is illustrated.