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.
We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca 2+ dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50 (Ca 2+ ) values for calmodulin binding and enzyme activation from the wild-type values of 180 ± 2 and 397 ± 23 nM to values of 109 ± 2 and 258 ± 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50 (Ca 2+ ) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 ± 4 and 118 ± 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50 (Ca 2+ ) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50 (Ca 2+ ) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca 2+ transient, while the reduction in EC 50 (Ca 2+ ) values is predicted to be the major contributor to enhancement at resting free Ca 2+ concentrations.The nitric oxide synthases catalyze formation of NO and L-citrulline from L-arginine and oxygen, with NADPH as the electron donor (1). The importance of NO generated by endothelial nitric oxide synthase (eNOS) 1 in the regulation of smooth muscle contractility is particularly well established and initially led to the discovery of its role in cell signaling (2). All of the synthase isozymes are functional homodimers of 130-160 kDa monomers that each contain a reductase and oxygenase domain (1). A significant difference between P450 reductase and the homologous reductase domains in eNOS and nNOS is the presence of autoinhibitory inserts in the latter (3,4). A CaM-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases require Ca 2+ and exogenous calmodulin (CaM) for activity (5,6).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (7-9). There are corresponding phosphorylation sites in human eNOS (7-9). Phosphorylation of the enzyme within the CaM-binding domain at Thr-497 blocks CaM
We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist-or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC 50 (Ca 2؉ ) The nitric-oxide synthases catalyze formation of NO and L-citrulline from L-arginine and O 2 , with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS) 2 in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130 -160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca 2ϩ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca 2ϩ ) 4 -CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca 2ϩ
The 45‐residue autoinhibitory insert in the endothelial nitric oxide synthase (eNOS) reductase domain inhibits electron transfer, and this effect is counteracted when CaM is bound to the enzyme. We have investigated the effects of phosphorylation at Ser‐617 and Ser‐635 in this region on the Ca2+ sensitivities of CaM binding and CaM‐dependent enzyme activation and on reductase and synthase activities. The mean EC50(Ca2+) values (± SD) for CaM binding to bovine eNOS and mutants with S617D, S635D, S617D/S635D substitutions or lacking the entire autoinhibitory insert were determined to be 220 ± 25, 109 ± 7, 202 ± 21, 117 ± 14, or 47 ± 9 nM, respectively. Corresponding mean EC50(Ca2+) values for eNOS activation were found to be 440 ± 55, 240 ± 28, 461 ± 56, 263 ± 28, or 124 ± 16 nM. The S617D substitution and deletion of the insert were found to increase CaM‐dependent synthase and cytochrome c reductase activities 2‐fold, and to respectively increase CaM‐independent reductase activity 3‐ and 6‐fold. The S635D substitution was found to have no significant effect on any property examined. These results indicate that phosphorylation at Ser‐617 acts synergistically with CaM to counteract the effects of the autoinhibitory insert. Given a resting free Ca2+ concentration in endothelial cells of 50 – 150 nM, phosphorylation at this site also appears to promote binding of CaM to the synthase under basal conditions.Source of research support: NIH Grant GM074887.
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