Bovine Endothelial Nitric Oxide Synthase
Created by Ziyi Gao
Bovine Endothelial Nitric Oxide Synthase (1nse) from Bos taurus is an enzyme that synthesizes nitric oxide, a key signaling molecule that regulates blood pressure, neurotransmission, and immune response. It achieves its function by a series of steps involving reduction of the heme iron, leading to O2 activation and subsequent oxidation of the L-Arg guanidine nitrogen atom to nitric oxide and L-citrulline (1). The first nitric oxide synthases were described in 1989, and the three major isoforms were purified between 1991 and 1994 (8). The molecular weight of eNOS is 99292.65 Da, and its isoelectric point is 6.37. The NOS family of proteins catalyzes the reaction as follows:
L-arg + 3/2 NADPH + H+ -> L-citrulline + 3/2 NADP + NO
Nitric oxide is also involved in smooth muscle relaxation via a cGMP-mediated signal transduction pathway. As a gaseous molecule, NO is a particularly useful second messenger because of its rapid diffusion amongst neighboring cells (3). NO will only be locally active due to its quick degradation. NO acts as a messenger by forming a complex with guanylate cyclase, and converts GTP into cyclic CMP. Furthermore, NO is a key molecule for vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and activates platelets in order to cause blood clotting. Overproduction of NO by iNOS and nNOS is linked to stroke and shock, whereas the production of NO by eNOS is integral to antiatherogenesis and angiogenesis. It is desired to produce an NOS inhibitor that will only inhibit iNOS and nNOS without touching the function of eNOS (1).
The structure of eNOS dominates its function and regulates its activity. The NOS family of proteins includes inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). nNOS and iNOS are found in the cell cytosol while eNOS is found on membranes. nNOS and eNOS are used by the cells to provide a steady source of dilute nitric oxide while iNOS is used when cells need to produce a large amount of NO in a reaction to pathogens (5). Endothelial nitric oxide synthase is a homodimer that is bundled together, exposing only 3000 A2 of surface area, of which 55% is nonpolar and 45% is polar (1). The primary structure of eNOS contains 1203 amino acids in its chain as opposed to 1434 amino acids in nNOS and 1153 amino acids in iNOS. The molecular weight of iNOS is less than eNOS and more for nNOS than eNOS. The secondary structure of this protein is made up of an assortment of alpha helices and beta sheets. The fold of the eNOS dimer is very similar to the mouse iNOS dimer at a lower resolution of 2.6 angstrom. Human eNOS and iNOS oxygenase domains (amino acids 82-508) are very similar in structure, its cofactors, and stereochemistry relative to the catalytic site (8). There are distinct genes for each type of NOS, but they have similar gene sequences that result in similarities in structure and primary structure. Their genetic similarity also carries implications regarding the evolution of their genes. Each monomer is composed of an N-terminal oxygenase domain and a C-terminal reductase domain (2). The function of eNOS relates mostly to the N-terminal domain where the heme prosthetic group is found and is required for dimerization. All three NOS molecules have to be in their dimer conformation in order to be catalytically active. This is because the monomer by itself cannot coordinate with Zn and thus cannot bind with H4B.
Crane et al. postulated from the crystal structure of the inactive iNOS monomer and dimer that pterin binding is useful for forming dimers, inducing conformational changes, and creating the L-Arg binding site, and enabling formation of the reductase and caveolin-binding sites (9). Further comparison of the H4B-bound and H4B-free forms of the heme domain by Raman et al. shows that it is not required for dimer formation nor is the pterin function useful for L-Arg binding and is not required for the active site. There was also no conformational difference in the protein at the pterin site, reductase or caveolin interaction sites. Furthermore, the disulfide bridge that Crane et al. observed from two cysteines turned out to be coordinating with a zinc ion, which stabilized the dimerization of eNOS (1).
The NOS family of proteins is regulated in vivo by calmodulin, a sensor protein that binds with Ca2+. While the C-terminal reductase module binds NADPH, flavin adenine dinucleotide (FAD), and flavin mononucleide (FMN), the N-terminal oxygenase module binds H4B, Fe-protoporphin IX, and L-Arg, calmodulin binds between the interfaces of these two modules. Calmodulin can activate nNOS to biosynthesize NO when bound with calcium ions. Calmodulin can thus be regulated by calcium concentrations and transduce the signal via NO as a second messenger. The process by which calmodulin mediates the electron transfer in NOS is poorly understood (6).
The associated ligands for eNOS (1nse) include glycerol, zinc ion, 5,6,7,8-tetrahydrobiopterin (H4B), ethylisothiourea, and protoporphyrin IX containing iron (the heme prosthetic group). The glycerol is primarily an artifact from protein purification located above the 4a position of the H4B ligand (1). The zinc ion plays a crucial role in stabilizing the binding site for H4B. It does so by coordinating with Cys-101 and Cys-96 in the N-domain of each subunit of eNOS. The reason for the coordination with zinc is largely because of the Cys-(X)4-Cys motif that is conserved among all NOS proteins. If the zinc ligand were absent, the strongly reducing conditions of the cytosol would prevent the sulfhydryl of the cysteine residues from forming a disulfide bond. Instead, the primary structure of this region would be disordered (1). Thus, the zinc ion coordinates with the cysteine residues in order to maintain the structure of the binding site for H4B. Another key feature of having ZnS4 in eNOS is its role in regulation of the amount of nitric oxide produced. If the cysteine ligands are nucleophilic, the ligands can undergo S-nitrosylation and release the zinc. If the zinc coordination is weak, then the function of eNOS will deteriorate and less NO will be synthesized. This type of feedback inhibition is used by nitric oxide to regulate NO biosynthesis in situ (1).
The key ligand that allows eNOS to form its activated dimer complex is 5,6,7,8-hydrotetrabiopterin, or H4B. This ligand shows novel pterin function, which is bound by H-bonding to the carboxylate oxygen of the heme propionate group. The high affinity between eNOS and pterin is a product of the H-bond network. Also note that the hydrogen bonds limit the conformation of the H4B to chiral centers at C6, C1’, and C2’ (1). This heme propionate group is also H-bonded to the amino group of the L-arg. The ethylisothiourea is another ligand, which can bind in place of H4B if it is not present. It is a potent inhibitor that also allows for tight binding of the L-arginine, showing similarity to the pterin function of H4B. Other mechanisms of H4B are inhibiting superoxide and hydrogen peroxide formation, modifying the heme environment, and protecting against inactivation. Although analogues have been able to replicate some features of H4B, such as stabilizing the dimer and modifying the heme environment from low to high spin state, they have not been able to affect NO biosynthesis (8). In the absence of H4B, superoxide formation by eNOS can lead to pathology.
As one would expect, FAD and FMN in the reductase domain act to transfer electrons from NADPH to the heme domain, similar to cytochrome p450. The flavin cofactors allow a two-electron donator (NADPH) to transfer an electron to a one-electron accepter by forming semiquinone radical intermediates. Electron flow appears to “cross over” from the flavin domain of one polypeptide chain to the heme domain of the other. This observation further cements the evidence that the NOS monomer is inactive and the dimer form must be required for its function (8).
The H4B cofactor stabilizes the positively charged pterin ring of L-Arg, making it specifically recognize the L-Arg in the H4B site. The stabilization does not occur due to protonation, but rather by restricting the lone pair on N-5 of the pterin ring. There is a possibility that reductase (FMN) may interact with the zinc center and pterin site and help in the reduction of the pterin radical to H4B. This radical has also been proposed in nNOS (1).
Hydrogen bonds form between L-Arg carboxyl group and Tyr-367 and the carboxyl group of Asn-376. Furthermore, the beta strand main chain hydrogen bonds include those formed by Sy-96 and Sy-101 with the peptide amino groups of Leu-102 and Gly-103, respectively. The amide nitrogen of Cys-101 has a hydrogen bond with the carbonyl functionality of Asn-468. The network of hydrogen bonds accounts for the negative charge on the sulfur ligands. Zinc is positioned near the heme groups and H4B in order to allow the pterin side chain hydroxyl to form a hydrogen bond with Ser-104. Val-106 contacts but does not bond with the pterin side chain. This network of interactions allows the H4B to interact with the heme and L-Arg and increases the protein’s stability and catalytic activity (1). This network also promotes the binding of H4B to the protein due to increased stability.
The crystallization and purification of the eNOS molecule lends itself to review as well because it may have a significant effect on how one might interpret the results of the crystal structure. The protein of interest was obtained by lysis via trypsin of a holo eNOS construct without 75% of the calmodulin-binding region. The crystals were flash frozen in liquid nitrogen and protein stabilization cocktail composed of glycerol, trehalose, mannitol, and sucrose was used as a cryoprotect. This could be the source of the glycerol shown as a ligand. The crystal structure was elucidated at 1.9 and 1.95 angstrom resolution to gain a better understanding of the structure.
Comparison of eNOS pterin interaction with pterin affinity for other enzymes show that eNOS uses the carboxylate function for pterin recognition. The H4B-binding site in eNOS is similar to other enzymes that use pterin as a substrate. A similar protein to the protein of interest is inducible nitric oxide synthase, which is also in the NOS family of proteins. Inducible nitric oxide synthase (1m8e) from Mus musculus functions almost exactly the same way as eNOS (6nse). The Z-score from the Dali server is 56.2, showing that the protein has very similar folds and the E value from the PSI-blast was 0.0, signifying that these two proteins have nearly the same primary structure. The key difference is that 7-nitroindazole in the iNOS is a bicyclic aromatic inhibitor, which creates a conformation change at Glu-371, thus the active site differs from eNOS in its molecular recognition properties (2). In addition, the mouse iNOS protein did not show the characteristic of cysteine residues in coordination with a zinc ion. Another very similar protein to bovine eNOS is nNOS from homo sapiens. With a Z-score of 58.1 and E-value of 0.0, this protein is like iNOS in that is has similar folds with eNOS and almost the same amino acid sequence. Outside of the NOS family, no proteins had a Z-score greater than 2.0 or an E-value less than 0.05 to compare.
Some particularly important residues are Cys-96 and Cys-101. Both of these residues are present on each subunit and two pairs of these two residues act to coordinate the zinc metal and thus stabilize the binding site for hydrotetrabiopterin. Regulation of the NOS enzymes by drug interactions is much sought after due to the variety of functions and pathologies that NO is part of in the body. The isoform inhibitors are key molecules in pursuing a drug to control NO biosynthesis. In one paper, it is believed that compounds with structural similarity to L-arg will bind to NOS as such and cause conformational changes that will inactivate NOS (6). However, no drug interaction has yet been created based on this.
Another group has created NOS inhibitors based on preserving the pteridine nucleus, much like H4B. By changing the substitution patterns in the 2,4,5,6,and 7 position of 4-oxo-pteridine nucleus, the molecules can act as effective NOS inhibitors (7). One modulator that slightly inhibits the activity of eNOS is called NOSIP, or eNOS interaction protein. This protein promotes translocation of eNOS from the membrane to intracellular sites, thus rendering it inactive. Both specificity and effectiveness have been observed in cell, but not necessarily in vitro due to presence of detergent in cell extract (10).