PEP-19 modulates calcium binding to calmodulin by electrostatic steering

Abstract

PEP-19 is a small protein that increases the rates of Ca²⁺ binding to the C-domain of calmodulin (CaM) by an unknown mechanism. Although an IQ motif promotes binding to CaM, an acidic sequence in PEP-19 is required to modulate Ca²⁺ binding and to sensitize HeLa cells to ATP-induced Ca²⁺ release. Here, we report the NMR solution structure of a complex between PEP-19 and the C-domain of apo CaM. The acidic sequence of PEP-19 associates between helices E and F of CaM via hydrophobic interactions. This allows the acidic side chains in PEP-19 to extend toward the solvent and form a negatively charged surface that resembles a catcher's mitt near Ca²⁺ binding loop III of CaM. The topology and gradients of negative electrostatic surface potential support a mechanism by which PEP-19 increases the rate of Ca²⁺ binding to the C-domain of CaM by 'catching' and electrostatically steering Ca²⁺ to site III.

Figure 1. Structural transitions in PEP-19 on binding to apo C-CaM are restricted to its acidic sequence and the core IQ motif.

(a) Backbone amide chemical shift changes calculated using equation (1) in ¹⁵N-labelled PEP-19 on binding to apo C-CaM. (b) Steady-state ¹⁵N{¹H} NOEs of ¹⁵N free PEP-19 (open circles) and ¹⁵N PEP-19 bound to apo C-CaM (closed circles). The ¹⁵N{¹H} NOE values and the uncertainty of NOE values (error bars) were determined as described in the ‘Methods' section.

Figure 2. Three-dimensional solution structure of the PEP-19:apo C-CaM complex.

(a) An stereo representation of an overlay of all backbone atoms for the final ensemble of 20 lowest energy conformers. Apo C-CaM is shown in dark blue. The disordered N terminus (aa 1–29), acidic sequence (aa 30–40) and IQ motif (aa 41–62) are shown in gold, red and light blue, respectively. (b) Ribbon representations for the structured regions of average minimized structure excluding the disordered N-terminal region of PEP-19. The colour scheme is the same as in a. Roman numerals III and IV indicate the Ca²⁺ binding loops of EF-hands III and IV, respectively, which are shown in yellow. All other figures also use the averaged minimized structure for the ensemble.

Figure 3. Interactions between the helical portion of PEP-19 and apo C-CaM.

(a) Intermolecular NOEs observed between residues in the helical segment of PEP-19 and residues in apo C-CaM. (b) A ribbon diagram of the helical portion of PEP-19 (light blue) with side chains (red) that form hydrophobic interactions with residues in apo C-CaM (grey).

Figure 4. Interactions between the acidic sequence of PEP-19 and helices E and F of Ca²⁺ binding site III in apo C-CaM.

The acidic sequence is shown in red, helices E and F in C-CaM are shown in dark blue, and Ca²⁺ binding loop III is in yellow. (a) Phe30, Ile32 and Met34 in the extended coil region in PEP-19 form hydrophobic interactions with residues in C-CaM (grey) that are at the interface between helices E and F in Ca²⁺ binding site III. (b) Selected planes acquired from F₁-filtered, F₃-edited NOESY-HSQC using ¹³C, ¹⁵N PEP-19 bound to unlabelled C-CaM. The planes highlight numerous intermolecular NOEs from Hδ1 of Ile32 and Hɛ of Met34 in PEP-19 to residues in C-CaM.

Figure 5. The acidic sequence of PEP-19 greatly increases the negative ESP near Ca²⁺ binding site III of apo C-CaM.

(a,b) Ribbon diagrams for the PEP-19:apo C-CaM complex and free C-CaM, respectively. Dark blue is C-CaM, yellow is Ca²⁺ binding loop III, red and light blue are the acidic sequence and core IQ motif in PEP-19, respectively. (c,d) Solvent excluded surfaces that are coloured based on ESP.

Figure 6. Mutation of Pro37 to Gly in PEP-19 increases the distance between the acidic sequence and Ca²⁺ binding site III in apo C-CaM.

Asp31 in PEP-19 and PEP(P37G) was mutated to Cys and spin labelled for PRE analysis as described in the methods. The grey bars in (a) the normalized amide I_ox/I_red intensity ratios derived from ¹H, ¹⁵N HSQC spectrum of apo C-CaM when bound to PEP-19(SL). The red and blue vertical lines indicate an increase or decrease, respectively, in I_ox/I_red due to mutation of Pro37 to Gly. The I_ox/I_red could not be calculated for residues indicated by (−) due to spectral overlap. Amide cross peaks for residue indicated by (*) are line broadened beyond detection when C-CaM is bound to PEP-19(SL), while those indicated by (x) are line broadened beyond detection when bound to PEP(P37G)SL. (b) The location of amides (small balls) for residues in apo C-CaM that show the greatest increase in distance from the PROXYL probe with an increase in I_ox/I_red of greater than 0.2 due to mutation of Pro37 to Gly. Calcium binding loop III is shown in yellow.

Figure 7. Steering of Ca²⁺ to Asp93 in Ca²⁺ binding loop III.

The topological features (channel and basin) near Ca²⁺ binding site III in the PEP-19:apo C-CaM complex are outlined by the dashed line. The surface contributed by Asp93 in C-CaM is shown as transparent, and is located in negatively charged basin. The green arrows illustrate funneling of Ca²⁺ to Asp93 guided by electrostatic steering and surface topology.

Figure 8. Comparison of acidic sequences in PEP-19 and Ng.

Blue boxes indicate residues that anchor the acidic sequence of PEP-19 to Ca²⁺ binding site III. Arrowheads indicate the position of acidic residues in the acidic sequences of PEP-19 and Ng.