Molecular tuning of an EF-hand-like calcium binding loop. Contributions of the coordinating side chain at loop position 3

Abstract

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

Figure 1

(A) The model EF-loop of GBP, including a comparison to a canonical EF-loop. Shown in stereo are the superimposed nine-residue Ca²⁺ binding loop backbones of GBP (solid bonds ; Vyas et al., 1987) and EF-hand site IV of human calmodulin (fine bonds; Chattopadhyaya et al., 1992). Also depicted are the coordinating side chains for each site, and, for the GBP site, the coordinating oxygens (small spheres) and bound Ca²⁺ ion (large sphere). The external glutamates of the two sites (n), which provide bidentate coordination in each site, originate from different sequence positions but display the same spatial location. In the GBP site, as in some canonical EF-hand sites (reviewed by Drake and Falke, 1996), the side chain at EF-loop position nine is a Gln or Glu residue that provides direct Ca²⁺ coordination at the solvent-facing axial position. By contrast, many canonical sites, including site IV of calmodulin, exhibit coordination by a solvent oxygen (not shown) at this position. Overall, the observed differences between the GBP loop and CaM loop IV fall within the range of differences observed between canonical EF-hand loops. (B) Crystal structures illustrating the three most common types of Ca²⁺ coordination by the third loop position. Shown are CPK representations of the bound Ca²⁺ ion (center), the surrounding seven oxygens forming the pentagonal bipyramidal coordination array (medium gray), and the position 3 side chain (carbons are light gray, nitrogen is dark gray). Position 3 coordination by Asn is illustrated by the galactose binding protein (GBP ; Vyas et al., 1987); Asp is exemplified by site I of parvalbumin (Parv; Kumar et al., 1990), and Ser is represented by site III of sarcoplasmic Ca²⁺ binding protein (Sarc ; Cook et al., 1993). (In the Sarc structure, the water molecule providing coordination at the upper axial position is not present in the crystallographic coordinates.)

Figure 1

(A) The model EF-loop of GBP, including a comparison to a canonical EF-loop. Shown in stereo are the superimposed nine-residue Ca²⁺ binding loop backbones of GBP (solid bonds ; Vyas et al., 1987) and EF-hand site IV of human calmodulin (fine bonds; Chattopadhyaya et al., 1992). Also depicted are the coordinating side chains for each site, and, for the GBP site, the coordinating oxygens (small spheres) and bound Ca²⁺ ion (large sphere). The external glutamates of the two sites (n), which provide bidentate coordination in each site, originate from different sequence positions but display the same spatial location. In the GBP site, as in some canonical EF-hand sites (reviewed by Drake and Falke, 1996), the side chain at EF-loop position nine is a Gln or Glu residue that provides direct Ca²⁺ coordination at the solvent-facing axial position. By contrast, many canonical sites, including site IV of calmodulin, exhibit coordination by a solvent oxygen (not shown) at this position. Overall, the observed differences between the GBP loop and CaM loop IV fall within the range of differences observed between canonical EF-hand loops. (B) Crystal structures illustrating the three most common types of Ca²⁺ coordination by the third loop position. Shown are CPK representations of the bound Ca²⁺ ion (center), the surrounding seven oxygens forming the pentagonal bipyramidal coordination array (medium gray), and the position 3 side chain (carbons are light gray, nitrogen is dark gray). Position 3 coordination by Asn is illustrated by the galactose binding protein (GBP ; Vyas et al., 1987); Asp is exemplified by site I of parvalbumin (Parv; Kumar et al., 1990), and Ser is represented by site III of sarcoplasmic Ca²⁺ binding protein (Sarc ; Cook et al., 1993). (In the Sarc structure, the water molecule providing coordination at the upper axial position is not present in the crystallographic coordinates.)

Figure 2

Binding free energy as a function of divalent cation size: effect of side chain substitutions at position 3 of the GBP Ca²⁺ binding loop. Plotted against effective ionic radius are binding free energies for divalent cations of group IIa. Each panel shows data for the native site (open circles, fine curve) and, excepting the first panel, for a site possessing an engineered side chain at the third loop position (filled circles, bold curve). The binding free energies were calculated as ΔG^o = RT ln(K _D), while effective ionic radii are those of Shannon (1976) for sevenfold coordination. Group IIa cations used were, in order of increasing radius: Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺. Dashed curves indicate regions of the free energy profile which were incompletely determined by the available radii. Lower limits (up arrows) indicate ions which yielded less than 50% displacement of Tb³⁺ at their maximum attainable concentrations in the Tb³⁺ competition assay (see materials and methods). An error bar is indicated for the largest error in each data set, calculated as RT ln(K _D ± 1 standard deviation). All measurements were at 25°C with 2.5 μM protein, 100 mM KCl, and 10 mM PIPES, pH 6.0.

Figure 3

Binding free energy as a function of trivalent cation size: effect of side chain substitutions at position 3 of the GBP Ca²⁺ binding loop. Legend as for Fig. 2, except that the indicated binding free energies are for trivalent cations from group IIIa (triangles) and the lanthanides (circles). In order of increasing radius, these were: Sc³⁺, Lu³⁺, Yb³⁺, Tm³⁺, Er³⁺, Ho³⁺, Y³⁺, Dy³⁺, Tb³⁺, Gd³⁺, Eu³⁺, Sm³⁺, Nd³⁺, Pr³⁺, Ce³⁺, and La³⁺.

Figure 4

Schematic structure illustrating alternate Ca²⁺ dissociation pathways in the galactose binding protein. Essentially the same picture can be proposed for Ca²⁺ dissociation from EF-hand sites (Drake and Falke, 1996). The gateway model hypothesizes that the preferred route to solvent is the axial pathway controlled by the gateway side chain at the ninth position of the Ca²⁺ binding loop; dissociation via this pathway yields upward movement of the bound metal ion toward solvent. The alternative dissociation pathway, leading to the right past the side chain at loop position 3, provides slower dissociation and is thus less important.