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Communication between the Zinc and Tetrahydrobiopterin Binding Sites in Nitric Oxide Synthase

The nitric oxide synthase (NOS) dimer is stabilized by a Zn2+ ion coordinated to four symmetry-related Cys residues exactly along the dimer 2-fold axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is ∼15 Å from the Zn2+. We have determined the crystal structures of the bovine endothelial NOS dimer oxygenase domain bound to three different pterin analogues, which reveal an intimate structural communication between the H4B and Zn2+ sites. The binding of one of these compounds, 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)-pteridinone (1), to the pterin site and Zn2+ binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn2+ binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn2+ site. Addition of excess Zn2+ stabilizes the Zn2+ site at the expense of weakened binding of 1. The unique structural features of 1 that disrupt the dimer interface are extra methyl groups that extend into the dimer interface and force a slight opening of the dimer, thus resulting in disruption of the Zn2+ site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn2+, pterin, and substrate binding sites.

M ammalian nitric oxide synthases (NOSs) require the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H 4 B) 1 to convert L-arginine to L-citrulline and nitric oxide, 2,3 an important second-messenger molecule in neural and cardiovascular systems. 4 The mammalian NOS enzyme family consists of three isoforms, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). 5 Each isoform is active only as a homodimer because the pterin binding site is located right at the dimer interface and monomeric NOS does not bind H 4 B or the substrate. 6 The dimer interface is formed between two N-terminal heme binding oxygenase domains that is further stabilized by the coordination of a Zn 2+ ion ligated to two cysteine thiols from each subunit (ZnS 4 ) 7,8 ( Figure 1). H 4 B plays the role of a redox active one-electron donor that activates the heme-bound O 2 , resulting in the formation of an H 4 B radical. 9 With L-Arg as the substrate, this radical is then rereduced by obtaining an electron from the ferrous NO complex generated at the end of the catalytic reaction, thus allowing the release of NO from the ferric heme. 10,11 All NOS isoforms share a strikingly similar pterin binding pocket with comparable H 4 B binding affinities, and cofactor and substrate binding events have been shown to synergistically stabilize the NOS dimer. 12−14 Low-temperature sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and urea dissociation studies indicate that the relative dimer strengths of the three mammalian NOS isoforms decrease from eNOS to nNOS to iNOS and that the role of H 4 B in dimer stability is less critical in eNOS. 15,16 The structural basis for this disparity, however, is not yet fully understood.
One way of exploring the relationship between dimer stability and H 4 B binding is to investigate the pterin binding . The Zn 2+ binding site is located at the dimer interface and ∼15 Å from the center of the pterin binding pocket in both molecules A and B of the dimer. Chain A is colored green, chain B yellow, pterin blue, and heme orange. All structural figures were prepared with PyMol (http://www.pymol.org). pocket using various pterin analogues. Moreover, inactive pterin analogues could potentially serve as NOS inhibitors 12,17−19 and have proven to be useful in probing the function of H 4 B. 20−23 In this study, we have determined the crystal structures of three novel pterin compounds ( Figure 2) analogous to H 4 B bound to eNOS, which has unexpectedly provided important insights into the intimate connection among the Zn 2+ , pterin, and substrate binding sites.

■ MATERIALS AND METHODS
Protein Expression and Purification. The bovine holo-eNOS pCWori construct containing an Amp R gene and an Nterminal six-His tag was expressed in Escherichia coli BL21-(DE3) cells already containing the calmodulin plasmid (with a Chl R gene), and the cells were then plated on LB agar with ampicillin (100 μg/mL) and chloramphenicol (35 μg/mL). E. coli cells inherently lack the machinery to synthesize H 4 B, ensuring that eNOS would remain free of H 4 B. A single colony was used to inoculate each 5 mL of LB starter culture (100 μg/ mL ampicillin and 35 μg/mL chloramphenicol). The culture was incubated for 8 h at 37°C with 220 rpm agitation. Each liter of TB medium (100 μg/mL ampicillin and 35 μg/mL chloramphenicol) was inoculated with 2 mL of LB starter culture. The cells were grown at 37°C with agitation at 220 rpm until the OD (600 nm) reached 1.0−1.2. Protein expression was then induced by adding 0.5 mM isopropyl β-D-thiogalactoside and 0.4 mM δ-aminolevulinic acid. New doses of ampicillin and chloramphenicol were also added, and cell growth resumed at 25°C and 100 rpm for 24 h. The cells were then harvested by centrifugation and stored at −80°C.
Cells were thawed and resuspended by being stirred for 3 h at 4°C in buffer A [50 mM sodium phosphate (pH 7.8), 10% glycerol, 0.5 mM L-Arg, 5 mM β-mercaptoethanol, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), and 200 mM NaCl]. The following protease inhibitors were added to buffer A before lysis: trypsin inhibitor (5 μg/mL), pepstatin A (1 μg/mL), and leupeptin (1 μg/mL). Cells were lysed by being passed through a microfluidizer at 18K psi (Microfluidics International Co.). The soluble fraction was isolated by centrifugation at 17000 rpm and 4°C for 1 h. The crude extract was then loaded onto a Ni 2+ -nitrilotriacetate column pre-equilibrated with 10 bed volumes of buffer A. After being loaded with the crude extract, the column was washed with 10 bed volumes of 10 mM imidazole in buffer A before being eluted with a 10 to 200 mM imidazole linear gradient in buffer A. Colored fractions were pooled and loaded onto a 2′,5′-ADP Sepharose column preequilibrated with buffer B [50 mM Tris-HCl (pH 7.8), 10% glycerol, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 0.5 mM L-Arg, and 200 mM NaCl]. The column was then washed with 10 bed volumes of buffer B and eluted with 10 mM NADP + in buffer B. Colored fractions were pooled and concentrated in a 30000 molecular weight cutoff (MWCO) Amicon concentrator at 4°C. The eNOS heme domain used for crystallization was generated by limited trypsinolysis: a 20:1 eNOS:trypsin weight ratio was used for a 1 h incubation at 25°C. The digested sample was then loaded onto a Superdex 200 column (HiLoad 26/60, GE Healthcare) controlled by an FPLC system and preequilibrated with buffer B to separate the heme domain and flavin-containing fragment generated by the trypsin digest. Fractions were pooled according to an A 280 /A 395 spectral ratio of <1.7, and sample homogeneity was determined by SDS− PAGE.
Synthesis of H 2 B Analogues. The preparation of pterin compounds 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)pteridinone (1 in Figure 2), 2-amino-9a-methyl-6,7,8,9,9a,10hexahydrobenzo[g]pteridin-4(3H)-one (2), and 2-amino-9amethyl-8,9,9a,10-tetrahydrobenzo[g]pteridine-4,6(3H,7H)dione (3) used in this study has been previously described. 24,25 Crystal Preparation. All eNOS heme domain samples were prepared for crystallization by being concentrated to 12 mg/mL in buffer B using a 30000 MWCO Amicon concentrator. Cocrystallization was conducted by combining protein with 5 mM cofactor and 5 mM L-arginine. Crystals were grown at 4°C in 18−20% PEG 3350 (w/v), 250 mM magnesium acetate, 100 mM cacodylate (pH 6.25), and 5 mM tris(2-carboxyethyl)phosphine (TCEP) in a sitting-drop vapor diffusion setup. Freshly grown crystals were passed stepwise through a cryoprotectant solution containing 20% PEG 3350, before being flash-cooled with liquid nitrogen. X-ray Diffraction Data Collection, Processing, and Structure Refinement. Cryogenic (100 K) X-ray diffraction data were collected remotely at Stanford Synchrotron Radiation Lightsource (SSRL) using the data collection control software Blu-Ice 26 and a crystal mounting robot. An ADSC Q315r CCD detector at beamline 7-1 or a Mar325 CCD detector at beamline 9-2 was used for data collection. Raw data frames were indexed, integrated, and scaled using HKL2000. 27 The binding of H 4 B cofactors was detected by the initial difference Fourier maps calculated with REFMAC. 28 The pterin molecules were then modeled in COOT 29 and refined using REFMAC. Water molecules were added in REFMAC and    Figure 2, the three dihydropterin analogues retain the ring structure of H 4 B and introduce variations only in the side chain. Figure 3 shows the electron density of the three dihydropterin analogues bound to eNOS. Compound 1 ( Figure 3A) exhibits the strongest and most welldefined electron density. As expected from its structural similarity to H 4 B, compound 1 fits into the pterin binding pocket quite well, maintaining most of the interactions found with H 4 B: the π−π stacking with W449, the H-bonds from its 2-aminopyrimidine nitrogens to the heme propionate A, the Hbond from O4 to R367, and the van der Waals contacts with aromatic residues of the other subunits (W76 and F462). Therefore, it is not surprising that the tetrahydro form of 1 can support the conversion of L-arginine to NO in nNOS. 31 From kinetic studies, the estimated K D for 1 is 115 μM compared to a value of 1.1 μM for H 4 B. 31 However, the two additional H-bonds from the dihydroxypropyl side chain of H 4 B to the carbonyls of S104 and F462 are lost in 1. Unique to 1 is the close contact from one of its methyl groups at the C7 position to the W447 side chain of the other subunit. Both 2 and 3 in the dihydro oxidation state introduce a third cyclohexane ring to replace the H 4 B side chain. Although the third ring is tolerated by the pterin binding pocket, the extra methyl group at C7 may generate steric clashes with the protein. As a result, 2 ( Figure 3B) and 3 ( Figure 3C) exhibit weaker electron density. Crystals of the eNOS−3 complex diffract poorly, which we have found correlates well with poor ligand binding. Because 3 is structurally similar to 2 and differs only in the carbonyl O atom on the cyclohexane ring, the additional steric crowding of this oxygen in 3 very likely accounts for why this pterin analogue binds more poorly. Overall, the inability of the three pterin analogues to form hydrogen bonds with S104 and F462 and the steric clashes from their protruding methyl groups may be attributed to a binding affinity that is poorer than that of the native pterin, H 4 B.
Disruption of the Zn 2+ Binding Site. As shown in Figure  4, a single Zn 2+ ion is situated at the dimer interface where it is tetrahedrally coordinated by symmetry-related Cys residues along the dimer axis. The Zn 2+ is ∼15 Å from the center of the pterin binding pocket in both subunits A and B of the dimer. Quite unexpectedly, we found that the binding of 1 completely disrupts the Zn 2+ binding region, as evidenced by a total lack of electron density for residues 91−109 and the Zn 2+ ( Figure 4A). It has been known for some time that both Zn 2+ and H 4 B contribute to dimer stability, 8,32,33 but this is the first indication that there is a relatively long-range communication between these two sites. To probe whether the binding of 1 and the binding of Zn 2+ are mutually exclusive, we soaked crystals of the eNOS−1 complex in a cryoprotectant solution supplemented with 50 μM Zn acetate. The crystal structure shows that Zn 2+ binding is restored ( Figure 4D), while the electron density for 1 and the substrate, L-Arg, are poorly defined ( Figure 3D). We next soaked crystals at a more moderate Zn acetate concentration of 20 μM. In this case, 1 and L-Arg bind well but the Zn 2+ site is disordered (data not shown). In contrast, binding of 2 and binding of 3 do not interfere with Zn 2+ binding ( Figure 4B,C). The interdependence of pterin and L-Arg binding is well-known and is very likely mediated by the fact H 4 B H-bonds to the same heme propionate as the α-amino group of L-Arg (Figure 3). 12−14 How Does the Pterin Site Communicate with the Zn 2+ Site? These results clearly show that there is a strong longrange communication between the Zn 2+ , pterin, and substrate binding sites. To explain why, we next conducted a detailed comparison between the eNOS−1 complex with a disordered Zn 2+ and the fully ordered eNOS structure with H 4 B bound. By superposition of the α-carbon backbone of chains A ( Figure  5B), we see that chain B has shifted away by a significant distance. For comparison, we next superposed H 4 B-bound to H 4 B-free eNOS structures ( Figure 5A) and did the same for bound structures of 2 ( Figure 5C) and 3 ( Figure 5D). In these cases, chain B does not move significantly relative to chain A. For the sake of consistency, we superposed the α-carbon backbone of chain B and observed a similar shift in chain A. To further quantify the observation, we calculated the root-meansquare deviations (rmsds) of chains A and B for residues 121− 482 in each pair of structures. For the eNOS−1 complex superposed on the basis of only chain A with an H 4 B-bound eNOS reference structure (PDB entry 9NSE), the rmsd of chain B is 1.23 Å, compared to the values of 0.27−0.44 Å calculated with the same method against the same reference structure for either an H 4 B-free structure or other pterin analogue structures that do not disrupt the Zn 2+ site ( Table 2).
A close examination of the eNOS−1 structure reveals that chain B moves away from chain A because of the methyl groups on C7 of 1 ( Figure 6B). Accommodating these methyl substituents forces W447 of chain B to shift away from chain A. The shift of W447 is not merely absorbed locally, but instead induces a global change in the entire chain B as a rigid body. The repulsion between the methyl group of 1 and W447 occurs in a symmetrical manner in both subunits, resulting in the slight opening of the dimer interface. The widened dimer interface causes disruption of key backbone contacts between the Cysbearing Zn 2+ binding hairpin fragment (residues 95−102) that includes contacts between C101 and N468 ( Figure 6B). Loss of those key interactions leads to the total disordering of the Zn 2+ binding site. Comparatively, the H 4 B-free eNOS structure ( Figure 6A) and those in complexes with 2 ( Figure 6C) and 3 ( Figure 6D) show practically no shift of W447. The extra methyl group in 2 and 3 is not directly pointing to W477, causing no disruption of the dimer interface. In the same way, we also compared the H 4 B-bound eNOS structure to the structure of the eNOS−1 complex supplemented with 50 μM Zn 2+ giving a restored Zn 2+ site, and the resulting superposition shows a tighter dimer ( Figure 7A) and a restored W447 position ( Figure 7B) at the expense of weakened binding of 1 ( Figure 3D). It also should be noted that residues directly contacting H 4 B, W447, W449, and F462, are conserved in both nNOS and eNOS.
What Does This Communication Reveal about the NOS Dimer? These results reveal that the eNOS dimer is able to loosen up and expand to accommodate 1. The side effect is that all hydrogen bonding interactions that stabilize the Zn 2+ binding site, mainly the two between N468 and C101, are weakened or lost, as shown in Figure 6. This effect being compounded on both chains causes a complete disruption of Zn 2+ binding and destabilization of the dimer.
Previous studies have shown that eNOS has the most stable dimer compared to nNOS and iNOS. 16 To obtain NOS−pterin