Calmodulin Transduces Ca²⁺ Oscillations into Differential Regulation of Its Target Proteins

Diverse physiological processes are regulated differentially by Ca²⁺ oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca²⁺ binding affinities, and target exchange rates that depend on both target identity and Ca²⁺ occupancy. The binding dynamics among Ca²⁺, Mg²⁺, calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca²⁺-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca²⁺ transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin

Properties of <i>Pf</i>SpdS inhibitors.

<p>Properties of <i>Pf</i>SpdS inhibitors.</p

Proposed classification of SpdS inhibitors.

<p>Proposed classification of SpdS inhibitors.</p

Ligand binding to <i>Pf</i>SpdS.

<p>(A) Pathways for substrates, products, and inhibitors. The enzyme is represented schematically by a black outline indicating its binding sites and their conformational changes upon ligand binding: left, MTA cavity (triangle; fully formed in the free enzyme, unchanged with bound ligands); middle, central aminopropyl cavity (distorted circle in the free enzyme; becomes circular with bound ligands); right, putrescine site plus distal aminopropyl cavity (distorted oval in the free enzyme; becomes rectangular with bound ligands, some of which do not fill the distal cavity completely). Coloring of schematically represented compounds follows the colored boxes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.g002" target="_blank">Fig 2</a>: aminopropyl donor substrate and its product, beige; polyamine substrates and products, green; inhibitors, blue. The double arrows show binding equilibria between forms; the single arrows indicate enzymatic reaction. Letters and numbers under each shape correspond to the classes defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a> and depicted in panel B. (B) Classification of ligands. Upper left, the schematic representation of the active site introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.g001" target="_blank">Fig 1</a> and used here to identify ligand-binding locations. Lower left box, the indicated substrates and products are shown as stick cartoons placed in the appropriate active-site locations; right box, representation as in the left box showing the active-site positions of the compounds listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a>. Numbers and letters below each entry correspond to classes defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163442#pone.0163442.t003" target="_blank">Table 3</a> and represented in panel A. Placements in the active-site locations are based on the respective crystal structures except for S_2a* and S_2b*, which are artificially positioned in the structure with dcAdoMet only based on structures P_2a and P_2b with MTA, and I_2b*, which is artificially positioned in the structure with dcAdoMet based on structure I_3a of NACD with MTA.</p

Overall structure and active site of <i>Pf</i>SpdS.

<p>(A) Monomer architecture. The N-terminal beta-sheet domain is light blue; the C-terminal Rossmann fold domain is dark blue. The active site is indicated in a cleft between the two domains, marked by a stick model of bound MTA and putrescine (green, based on PDB ID: 4BP1). The gatekeeper loop spanning the active site is shown in orange; when ligands are bound it adopts a loop-3₁₀ helix-loop structure that is approximated in the representation shown. (B) Active site. Labeled yellow shaded or outlined oval shapes of different sizes represent the indicated parts of the active site referred to in the text. The larger dcAdoMet site (black-outlined oval at right) is conceptually divided into an MTA cavity (large upper shaded oval) and a central aminopropyl cavity (small lower shaded oval). The putrescine site (central black-outlined oval) is adjacent to a distal aminopropyl cavity (shaded oval at upper left). The substrates dcAdoMet and putrescine are represented as stick cartoons with green carbon atoms and other atoms in atomic colors (blue nitrogen, red oxygen, and yellow sulfur); these substrates do not occur together in any existing crystal structure because the enzyme reaction would occur. The cartoon is a composite based on separate structures with dcAdoMet and with MTA and putrescine.</p

Inhibition of <i>Pf</i>SpdS activity.

<p>(A) Lineweaver-Burke plot for 4MAN. Reciprocal velocity <i>vs</i>. reciprocal putrescine concentration at 0.1 mM dcAdoMet in presence of 4MAN at final concentrations of 0 μM, open circles (○); 20 μM, filled triangles (▲); 40 μM, open diamonds (◊); or 100 μM, filled circles (●). Solid lines are linear regression fits for each 4MAN concentration. Each symbol represents the average of two technical replicates from one independent measurement (range = < 17%). (B) Secondary plot for 4MAN. The slope of each line in panel A is plotted <i>vs</i>. 4MAN concentration. The solid line is the linear regression fit, yielding the K_i value of 8.2 μM from the abscissa intercept. (C) Lineweaver-Burke plot for AdoDATO. Reciprocal velocity <i>vs</i>. reciprocal putrescine concentration at 0.1 mM dcAdoMet in presence of AdoDATO at final concentrations of 0 μM, open circles (○); 5 μM, filled triangles (▲); 10 μM, open diamonds (◊); or 25 μM, filled circles (●). Solid lines are linear regression fits for each AdoDATO concentration. Each symbol represents the average of two technical replicates from one independent measurement (range = < 9%). Two independent measurements were performed with reproducible results. (D) Secondary plot for AdoDATO. The slope of each line in panel C is plotted <i>vs</i>. AdoDATO concentration. The solid line is the linear regression fit, yielding the K_i competitive value of 3.4 μM from the abscissa intercept.</p

Substrate binding sites. A. NADH.

<p>View of the active site with NADH bound in the optimized position found by docking as described in the text. Green, molecular surface of holoWrbA calculated from the 2.05 Å crystal structure (PDB ID 3B6J) after removal of the FMN cofactor. Oxidized FMN is depicted as a skeletal model in atomic colors with cyan carbon, and docked NADH with white carbons for differentiation from FMN. Dashed lines represent the indicated distances in Å between nicotinamide C4 and each indicated electron acceptor site of FMN. <b>B. Mutual exclusivity of NADH and BQ.</b> Viewpoint of the binding cavity as in panel A but slightly zoomed out to better depict the steric environment of the full pocket. Translucent white indicates the molecular surface of NADH in the position identified by docking as in panel A; red indicates the molecular surface of BQ calculated from the 1.99 Å crystal structure of the BQ/WrbA complex (PDB ID 3B6K). The part of each substrate that is occluded by the other is represented by the overlap between the red and translucent white surfaces.</p

Steady-state kinetics.

<p>Initial velocity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#s4" target="_blank">Methods</a>) is plotted <i>vs.</i> substrate concentration. <b>A.</b> NADH at constant [BQ] = 50 µM. <b>B.</b> BQ at constant [NADH] = 50 µM. <b>C.</b> DCPIP at constant [NADH] = 50 µM. Each plot depicts three temperature treatments of WrbA prior to assay (see text): squares, 5°C; triangles, 23°C; circles, 5°C after 23°C. Solid lines are intended only to guide the eye and do not represent fits to the data.</p

Sedimentation velocity.

<p>Each panel shows the sedimentation velocity profile using the whole boundary g(s*) approach of Stafford <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone.0043902-Stafford1" target="_blank">[28]</a> for apoWrbA (black), WrbA+50 µM FMN (red), and WrbA+50 µM FMN+0.5 mM NAD (blue). A, 3 µM total protein (monomer) at 5°C; B, 3 µM total protein (monomer) at 20°C; C, 20 µM total protein (monomer) at 5°C; D, 20 µM total protein (monomer) at 20°C.</p

Substrate affinity.

<p><b>A.</b> NADH binding to 50 µM apoWrbA determined by UV spectroscopy. Difference absorbance at 265 nm (see text) is plotted <i>vs.</i> [NADH]. The solid line is intended only to guide the eye and does not represent a fit to the data. <b>B.</b> NAD binding to 200 µM apoWrbA detected by ³¹P NMR. Spectra at 100, 200, 500, 1000 and 2000 µM NAD from bottom to top, respectively, are overlaid. The bracket with four arrows indicates the doublet pair characteristic of free NAD.</p