Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino- and carboxy-terminal halves

Abstract

Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

Fig. 1.

Amino acid sequence of CaVP including N-CaVP (arrow points to the last residue) and C-CaVP (arrow points to the first residue). EF-hand motifs are indicated in bold. Overlapping of both segments is emphasized in box. Relevant residues for this study are highlighted. Underlined lysines stand for TML. X, Y, and Z are the residues positioned at the vertices of the Ca²⁺-coordinating bipyramid.

Fig. 2.

Ca²⁺ binding to recombinant (open circles) and wild-type CaVP (closed circles), as well as C-CaVP (triangles). Ca²⁺ binding was measured by flow dialysis at 25°C in buffer C. The solid line represents the theoretical isotherm generated with the intrinsic binding constants 4.6 × 10⁶ and 7.4 × 10³ M⁻¹ for K′_Ca1 and K′_Ca2, respectively.

Fig. 3.

Near-UV CD spectra of C-CaVP (A), N-CaVP (B), and CaVP (C) at pH 7.5 in the presence of 5 mM CaCl₂ at different temperatures: 10°C (solid line), 90°C (dashed line), after heating to 90°C and cooling down to 10°C (dotted line). (D) Near-UV CD spectra of C-CaVP (dotted line), N-CaVP (dashed line), CaVP (solid line), and of the calculated sum of both segments (chain line).

Fig. 4.

Temperature dependence of the CD intensity for CaVP, N-CaVP, and C-CaVP at pH 7.5. (A) CaVP in the presence of 1 mM EGTA at 291 (thin dotted line) and 222 nm (thick dotted line) and in the presence of 2 mM CaCl₂ at 291 (thin solid line) and 222 nm (thick solid line). (B) N-CaVP at 222 nm (thick line) and 291 nm (thin line). (C) C-CaVP at 222 nm in the presence of 1 mM EGTA. The inset shows the first derivative of the CD melting curve.

Fig. 5.

Temperature dependence of the partial molar heat capacity of CaVP, N-CaVP, and C-CaVP at pH 7.5. (A) apo form of CaVP in the presence of 0.1 mM EGTA (dotted line), 1Ca²⁺ form in 0.1 mM CaCl₂ (solid line), and 2Ca²⁺ form in 2 mM CaCl₂ (dashed line). The inset shows the temperature dependence of the excess heat capacity for CaVP low temperature peak. (B) N-CaVP in the presence of 5 mM CaCl₂. (C) C-CaVP in the presence of 0.1 mM EGTA (dotted line), 0.1 mM CaCl₂ (solid line), and 2 mM CaCl₂ (dashed line).

Fig. 6.

Interaction analysis of N-CaVP, C-CaVP, and CaVP with CaVPT. (A) N-CaVP, C-CaVP, recombinant CaVP, and an equimolar mixture of N-CaVP, C-CaVP, or recombinant CaVP with CaVPT, in the presence of 2 M urea, were microdialyzed 60 min against buffer A containing 100 mM NaCl plus 1 mM CaCl₂, before loading on a native gel (12.5% acrylamide) in the presence of 10% glycerol. All samples contain 2.5 μg of each protein. Arrows indicate the mobility of the complex formed in the presence of Ca²⁺. Recombinant CaVP represents the positive control (same results with wild-type CaVP). This protein migrates in three bands. (B) Samples were chromatographed on a size-exclusion S-200 column under the same conditions as standards of known molecular masses (top), and analyzed by SDS-PAGE (15% acrylamide). An equimolar mixture of either C-CaVP and CaVPT (a), or of C-CaVP, CaVP, and CaVPT (d) was dialyzed against buffer A plus 100 mM NaCl and 1 mM CaCl₂. Addition of 2 mM EDTA dissociates the C-CaVP/CaVPT complex (b). C-CaVP alone has a tendency to dimerize in 1 mM CaCl₂ (c). At left, the positions of SDS-PAGE molecular markers are indicated (kD).