Intrinsically disordered PEP-19 confers unique dynamic properties to apo and calcium calmodulin

Abstract

PEP-19 (Purkinje cell protein 4) is an intrinsically disordered protein with an IQ calmodulin (CaM) binding motif. Expression of PEP-19 was recently shown to protect cells from apoptosis and cell death due to Ca(2+) overload. Our initial studies showed that PEP-19 causes novel and dramatic increases in the rates of association of Ca(2+) with and dissociation of Ca(2+) from the C-domain of CaM. The goal of this work was to study interactions between the C-domain of CaM (C-CaM) and PEP-19 by solution nuclear magnetic resonance (NMR) to identify mechanisms by which PEP-19 regulates binding of Ca(2+) to CaM. Our results show that PEP-19 causes a greater structural change in apo C-CaM than in Ca(2+)-C-CaM, and that the first Ca(2+) binds preferentially to site IV in the presence of PEP-19 with exchange characteristics that are consistent with a decrease in Ca(2+) binding cooperativity. Relatively weak binding of PEP-19 has distinct effects on chemical and conformational exchange on the microsecond to millisecond time scale. In apo C-CaM, PEP-19 binding causes a redistribution of residues that experience conformational exchange, leading to an increase in the number of residues around Ca(2+) binding site IV that undergo conformational exchange on the microsecond to millisecond time scale. This appears to be caused by an allosteric effect because these residues are not localized to the PEP-19 binding site. In contrast, PEP-19 increases the number of residues that exhibit conformational exchange in Ca(2+)-C-CaM. These residues are primarily localized to the PEP-19 binding site but also include Asp93 in site III. These results provide working models for the role of protein dynamics in the regulation of binding of Ca(2+) to CaM by PEP-19.

Figure 1. Effect of PEP-19 on C-CaM amides in the absence and presence of Ca²⁺

(A) Amide chemical shift perturbations (Δδ_avg) for Ca²⁺-bound C-CaM at 298 K are shown by black bars. Grey bars show Δδ_avg that result from binding PEP-19 to the C-domain of intact Ca²⁺-CaM at 320 K (14). Asterisks denote resonances with significant line-broadening in the presence of PEP-19. (B) Δδ_avg for apo C-CaM at 298 K. Panels (C) and (D) map residues with Δδ_avg >0.2 ppm on the solvent accessible surfaces of Ca²⁺-CaM (pdb:1J7P) and apo-C-CaM (pdb:1F71), respectively. The structures are presented in the same relative orientation. The diagram below panel B shows the relative positions of the helices E to H in the primary sequence of CaM. The 12 amino acids Ca²⁺ binding sites III and IV are also shown in grey. Coil, β-strand and α-helical portions of these structures are indicated by loops, arrows, and boxes, respectively.

Figure 2. Effect of PEP-19 on the Ca²⁺ binding constant (K_ca) and Hill coefficient (n)

Intrinsic tyrosine fluorescence was monitored during titration of CaM (○), CaM+PEP-19 (□), C-CaM (●), and C-CaM/PEP-19 (■) with Ca²⁺. The lines show fits of the data to the Hill equation (Eq.1).

Figure 3. Ca²⁺ -dependent changes in amide chemical shifts of C-CaM show strong cooperativity

Resonance of Thr117 in the linker region of C-CaM exhibited two-state slow exchange at various molar ratio of Ca²⁺ added into C-CaM, indicating highly cooperative Ca²⁺ binding to sites III and IV. (A) [Ca²⁺]/[C-CaM]=0; (B) [Ca²⁺]/[C-CaM]=0.3; (C) [Ca²⁺]/[C-CaM]=0.6; (D) [Ca²⁺]/[C-CaM]=2.0.

Figure 4. Ca²⁺ -dependent changes in amide chemical shifts of C-CaM in the presence of PEP-19 show decreased cooperativity

Various ratios of [Ca²⁺]/[C-CaM] between 0 and 2.0 are indicated. Exchange characteristics for Thr117 shown in Panel (A) indicate lower degree of cooperativity of Ca²⁺ binding in the presence of PEP-19. Exchange characteristics for Gly98 in Ca²⁺ binding site III and Gly134 in the Ca²⁺ binding site IV shown in Panel (B) indicates preferential binding of the 1^st Ca²⁺ to site IV.

Figure 5. Backbone ¹⁵N relaxation parameters for apo C-CaM, apo C-CaM/PEP-19, Ca²⁺-bound C-CaM and Ca²⁺-bound C-CaM/PEP-19

R₁, R₂, [¹H]-¹⁵N NOE, R₂R₁ products and R₂/R₁ ratios are shown for apo- and Ca²⁺-C-CaM in the absence (open circles) and presence (closed circles) of PEP-19. The dashed (with out PEP-19) and solid lines (with PEP-19) in Panels C, D, G and H indicate the mean +1.0 SD. Asterisks denote resonances with significant line-broadening in the presence of PEP-19.

Figure 6. Reduced spectral density mapping for apo C-CaM, apo C-CaM/PEP-19, Ca²⁺-bound C-CaM and Ca²⁺-bound C-CaM/PEP-19

Reduced spectral density parameters were calculated using Eq. (3) to Eq. (7). J(ω_H), J(ω_N), and J(0) are shown for apo- and Ca²⁺-C-CaM in the absence (open circles) and presence (closed circles) of PEP-19. The mean +1.0 SD of J(0) for apo and Ca²⁺-bound C-CaM in the absence and presence of PEP-19 is indicated by dash and solid line, respectively. Asterisks denote resonances with significant line-broadening in the presence of PEP-19.

Figure 7. Identification of residues exhibiting conformational exchange on the μs –ms timescale in PEP-19-bound apo C-CaM and Ca²⁺-C-CaM based on reduced spectral density mapping

Values on the Y-axis are calculated by (J(0)_i – J(0)_avg)/SD. Residues with (J(0)_i – J(0)_avg)/SD >1.0 are considered to undergo significant chemical/conformational exchange. Asterisks indicate residues with extreme line broadening.

Figure 8. Summary of residues that exhibit significant conformational exchange for apo and Ca²⁺-bound C-CaM in the presence of PEP-19

Panels (A) and (B) show the NMR solution structures for apo (pdb: 1F71) and Ca²⁺-bound C-domain of CaM (pdb: 1J7P), respectively. Colored balls indicate residues that exhibit significant conformational exchange, and are shown by bold lettering in Table 2.