Biphasic Ca2+-dependent switching in a calmodulin-IQ domain complex

Abstract

The relationship between the free Ca2+ concentration and the apparent dissociation constant for the complex between calmodulin (CaM) and the neuromodulin IQ domain consists of two phases. In the first phase, Ca2+ bound to the C-ter EF hand pair in CaM increases the Kd for the complex from the Ca2+-free value of 2.3 +/- 0.1 microM to a value of 14.4 +/- 1.3 microM. In the second phase, Ca2+ bound to the N-ter EF hand pair reduces the Kd for the complex to a value of 2.5 +/- 0.1 microM, reversing the effect of the first phase. Due to energy coupling effects associated with these phases, the mean dissociation constant for binding of Ca2+ to the C-ter EF hand pair is increased approximately 3-fold, from 1.8 +/- 0.1 to 5.1 +/- 0.7 microM, and the mean dissociation constant for binding of Ca2+ to the N-ter EF hand pair is decreased by the same factor, from 11.2 +/- 1.0 to 3.5 +/- 0.6 microM. These characteristics produce a bell-shaped relationship between the apparent dissociation constant for the complex and the free Ca2+ concentration, with a distance of 5-6 microM between the midpoints of the rising and falling phases. Release of CaM from the neuromodulin IQ domain therefore appears to be promoted over a relatively narrow range of free Ca2+ concentrations. Our results demonstrate that CaM-IQ domain complexes can function as biphasic Ca2+ switches through opposing effects of Ca2+ bound sequentially to the two EF hand pairs in CaM.

Fig. 1. Fluorescent reporter systems used to monitor interactions between CaM and the neuromodulin IQ domain. (A)

BSCaM_IQ consists of ECFP and EYFP variants of GFP joined by an IQ domain. When CaM is bound to this domain changes in the orientation and/or distance between the two fluorescent proteins decrease fluorescence resonance energy transfer (FRET) between the ECFP and EYFP components. Thus, under 430 nm excitation ECFP donor fluorescence, measured at 480 nm, is increased and EYFP acceptor fluorescence, measured at 525 nm, is decreased. (B) Complete fluorescence emission spectra for BSCaM_IQ under 430 nm excitation. The solid line is the emission spectrum determined in the absence of CaM, the dashed line is the spectrum determined in the presence of a saturating apoCaM concentration. The remaining spectra were determined in the presence of saturating concentrations of Ca²⁺-free NC_xCaM or N_xCCaM (□), Ca²⁺-saturated native CaM (●), N_xC₂CaM (○) and N₂C_xCaM (△). (C) A bipartite reporter system consisting of ECFP-CaM and EYFP-neuromodulin fusion proteins. As illustrated in the figure, formation of the complex between CaM and neuromodulin allows FRET between the ECFP and EYFP labels to occur under 430 nm excitation.

Fig. 2. Binding of CaM to BSCaM_IQ in the presence of 10 (◆) and 250 (□) μM free Ca²⁺, or 3 mM BAPTA (■)

The fractional change in 525 nm fluorescence produced by the decrease in FRET associated with formation of the complex is defined as (F_max−F)/(F_max−F_min), where F corresponds with the emission measured after each addition, and F_max and F_min correspond with the emissions of CaM-free and CaM-saturated BSCaM_IQ. A 50 μM BSCaM_IQ concentration was used for these experiments. The apparent K_d values determined from fits of these data to a simple one-site hyperbolic binding equation (not shown) are listed in Table 1.

Fig. 3. Binding of N_xCCaM or NC_xCaM to BSCaM_IQ

(A) Fractional changes in 525 nm fluorescence emission, defined as described in the legend to Fig. 2, associated with binding of N_xCCaM (□) or NC_xCaM (△) to 50 nM BSCaM_IQ under nominally Ca²⁺ free conditions (3 mM BAPTA). (B) Fractional changes in 525 nm fluorescence emission associated with binding of N_xCCaM (■) and NC_xCaM (▲) to 150 nM BSCaM_IQ at a free Ca²⁺ concentration of ~250 μM. Apparent K_d values derived from the data presented in panels A and B are listed in Table 1.

Fig. 4. Ca²⁺-dependent dissociation of the N_xCCaM or NC_xCaM complexes with BSCaM_IQ

Fractional changes in 525 nm fluorescence emission associated with formation of the complexes between 12.5 μM BSCaM_IQ and 1 μM N_xCCaM (◆) or NC_xCaM (■). All free Ca²⁺ concentrations were verified using indo-5F or mag-indo-1. Fractional changes in fluorescence are defined using an F_max value measured at the end of the experiment, which accounts for residual CaM binding in the presence of Ca²⁺. Otherwise fractional changes are defined as described in the legend to Fig. 2. Apparent Ca²⁺-binding constants derived from fits of these data to equation 2 are listed in Table 2.

Fig. 5. Direct measurements of Ca²⁺ binding to native and mutant CaMs in the presence and absence of BSCaM_IQ

(A) 263 nm absorbance data for 40 μM dibromo-BAPTA measured in the presence of 20 μM native CaM (■) or 20 μM native CaM and 100 μM BSCaM_IQ (□). (B) Absorbance data for 40 μM dibromo-BAPTA measured the presence of 20 μM N_xCCaM (●) or 20 μM N_xCCaM and 100 μM BSCaM_IQ (○). For presentation purposes these data have been normalized to the absorbance of Ca²⁺-free dibromo-BAPTA determimed at the completion of each titration experiment. Pooled data from 3 separate Ca²⁺ binding experiments are presented for each set of conditions examined. The dissociation constants derived from fits of these and similar data to equation 3 or 4 are listed in Table 2 (28).

Fig. 6. A general four-state scheme for Ca²⁺-dependent switching in a CaM-IQ domain complex

B indicates the bound IQ domain protein; N and C indicate the N-ter and C-ter EF hand pairs in CaM, with subscripts denoting their Ca²⁺-liganded states; the absence of a subscript indicates a Ca²⁺-free EF hand pair.

Fig. 7. Minimal scheme for Ca²⁺-dependent switching in the neuromodulin CaM-IQ domain complex

Ca²⁺ binding via the N-ter→C-ter pathway depicted in the general scheme appears to be strongly disfavored, so it can be omitted from the minimal scheme describing the Ca²⁺-dependence of the CaM-neuromodulin complex under steady-state conditions.

Fig. 8. The relationship between the free Ca²⁺-concentration and the dissociation constant for the complex between CaM and the neuromodulin IQ domain is bell shaped

All free Ca²⁺ concentrations were verified using indo-5F or mag-indo-1. (A) The fractional fluorescence response of 250 nM BSCaM_IQ in the presence of 20 μM CaM (B) The fractional fluorescence response of 5 μM BSCaM_IQ in the presence of 1 μM CaM (■), and the fractional response of a solution of 1 μM ECFP-CaM and 5 μM EYFP-neuromodulin (△). Because FRET increases when this complex is formed, the fractional response in this case defined as (F−F_min)/(F_max−F_min). The curves in both panels were calculated as described under “Materials and Methods” according to the scheme depicted in Fig. 7. The F_min/F_max values used to generate for these curves were adjusted slightly to achieve the fits shown. All adjusted values were within 3% of the mean values given in Table 1.

Fig. 9. Various species produced in a solution of neuromodulin (NM) and CaM as a function of the free Ca²⁺ concentration

Calculations were performed as described under “Materials and Methods” using CaM and NM concentrations of 10 μM based on the scheme depicted in Fig. 7. All calculated concentrations are expressed relative to the total CaM concentration. Curves correspond with the relative concentrations of bound NM (△), total free CaM (no symbol), free calci-CaM (○), NCCaM-NM complex (▲), NC₂CaM-NM complex (■), and N₂C₂CaM-NM complex (◆). Calci-CaM refers to the total of all free Ca²⁺-liganded CaM species, which primarily consist of NC₂CaM and N₂C₂-CaM. Otherwise, the nomenclature for the various Ca²⁺-liganded species of CaM is given in the legend to Table 1.