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Introduction

Endothelial Nitric Oxide Synthase (eNOS)^[1] is a major actor in the regulation of cardiovascular processes, since it induces the production of Nitric Oxide (NO) in vascular endothelial cells. NO is involved in several processes such as vessel vasodilatation, vascular smooth muscle cell proliferation, angiogenesis. The activity of eNOS is related to intracellular calcium concentration and its activation requires the calcium binding protein, CalModulin (CaM).

The structure of CaM protein bound to the CaM binding domain of eNOS has been obtained thanks to a solution NMR^[2], a nuclear magnetic resonance that enables the determination of structures but also interactions between molecules.

Structure and function

eNOS pathway and production of NO

Many stimuli such as vascular endothelial growth factor (VEGF), insulin, estrogen or bradykinin can lead to the production of NO by activated eNOS. The latter can be found in several membrane compartments : lipid rafts, plasma membrane and intracellular membranes such as the one of the Golgi complex.

The production of NO involves first the formation of Ca2+/CaM complex which binds to eNOS to the CaM binding domain situated between the oxygenase and the reductase domains, enhancing thus NO production by promoting an intramolecular electron transfer between FAD and FMN. eNOS contains an insert of 40-50 amino acids in the middle of the FMN binding domain. This insert is suspected to act as an auto-inhibitory loop that inactivates the enzyme when the concentration of Ca2+ in the cell is too low. Indeed, the physical displacement of this insert seems to be required so that Ca2+/CaM complex can access its binding domain. The binding of this complex is crucial since it allows the electron flow through the reductase domain of eNOS. Indeed, the electron transfer starts with a two-electron reduction of FAD by NADPH. Through the interaction between the FMN domain and the FAD domain, FMN accepts an electron. Then, the FMN domain donates an electron to the heme domain. Nevertheless, this electron transfer requires a conformational change so that the FMN domain can interact with the heme domain. Thus, in the oxygenase domain, the catalysis of the oxygen with L-arginine generates citrulline ad NO as products^[3].

Structure of CaM

Calmodulin is a 148-amino-acid peptide containing 2 symmetrical globular calcium domains connected by a flexible central linker region that is a 28-amino-acid-long alpha helix. The are the first calcium domain and the are the second one. CaM has 4 EF hand motifs (2 at each globular calcium binding domain) highly conserved among calcium binding proteins. EF hand motif is suitable for the binding of one calcium ion since an electronegative environment is established.

Structure of eNOS

eNOS is a homodimeric enzyme consisting of 2 main domains : a N-terminal oxygenase domain and a C-terminal reductase domain. The N-terminal oxygenase domain is composed of the heme and the tetrahydrobiopterin (BH4) binding sites and also the binding sites for the substrates L-arginine and oxygen, whereas the FMN binding subdomain and the FAD/NADPH binding subdomains are included in the C-terminal reductase. Those two domains are connected by a 17-amino-acid-long CaM binding domain which is essential for the efficient transfer of an electron from the reductase domain of one monomer to the heme domain of the adjacent monomer in order to produce NO^[4].

Interaction between CaM and the CaM binding domain peptide of eNOS

The 3D structure^[5] shown here represents the interaction between the and the .

In an antiparallel manner, the N-terminal helices I–IV and the C-terminal helices VI–VIII of CaM bind and wrap the CaM binding peptide of eNOS to interact with it. The eNOS peptide has an α-helical core composed of 12 residues from Phe496 to Ala507. The main interaction is the one between CaM and 4 hydrophobic residues of eNOS : Phe496, Ala500, Val503 and Leu509. Furthermore, the basic residues Arg492, Lys493 and Lys494 at the eNOS N-terminus bind through electrostatic forces to glutamate residues at the CaM termini.

Met76 on the central linker of CaM maintains the structure of its helix IV and interacts with Ile505 and Ser508 of eNOS. Met76 also associates with helix I (Phe12, Ala15, Phe68, Met72) at the CaM N-terminus which leads to helix I turning slightly. This helix shift of CaM counterbalances the negative charges of Glu498 on eNOS. Moreover, the Lys494 on eNOS balances, as a counterion, the highly negatively charged glutamates^[6][7].

The phosphorylation of Thr495 in eNOS CaM binding site inactivates eNOS since CaM cannot bind to its binding domain anymore. This threonine 495 residue is phosphorylated by several protein kinases such as protein kinase C, cyclic-nucleotide dependent protein kinase and AMP-activated protein kinase. The negative charged phosphate group of Thr495 generated by those protein kinases could induce an electrostatic repulsion with glutamate residues of CaM (Glu7 and Glu127)^[8].

Disease

As NO has a really important role in cardiovascular processes, a malfunction in the production of NO can contribute to diseases such as atherosclerosis, hypertension. Indeed, some risk factors such as high blood pressure, high glucose or lipids lead to the production of superoxide which interacts with NO. Thus, it produces peroxynitrite which interacts with DNA, proteins and lipids and this leads to cell necrosis or apoptosis. The interaction with NO also prevents NO from protecting the vasculature, such as inhibiting the proliferation of vascular smooth muscle cells, the platelet aggregation or the leukocyte adhesion to the endothelium. Cardiovascular diseases can be prevented thanks to the well functioning of eNOS and NO production.

References

1. ↑ eNOS signaling[1]
2. ↑ Solution NMR, Instruct Interacting Biology[2]
3. ↑ Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation, Brian C. Smith, Eric S. Underbakke, Daniel W. Kulp, William R. Schief and Michael A. Marletta[3]
4. ↑ Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation, Brian C. Smith, Eric S. Underbakke, Daniel W. Kulp, William R. Schief and Michael A. Marletta[4]
5. ↑ Structure from PDB[5]
6. ↑ Structural basis for endothelial nitric oxide synthase binding to calmodulin, Mika Aoyagi, Andrew S. Arvai, John A. Tainer and Elizabeth D. Getzoff[6]
7. ↑ PDBsum entry 2ll7 on EMBL-EBI[7]
8. ↑ Structural basis for endothelial nitric oxide synthase binding to calmodulin, Mika Aoyagi, Andrew S. Arvai, John A. Tainer and Elizabeth D. Getzoff[8]