Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase

Abstract

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Martásek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.

Figure 1

Two different binding orientations of cis-pyrrolidine compounds. (A) The aminopyridine of (3′S, 4′S)-2 interacts (dashed lines) with Glu592 while (B) the aminopyridine of (3′R, 4′R)-2 is flipped and interacts with heme propionate D. All of the structural figures were prepared with PyMOL (www.pymol.org).

Figure 2

Display of the anomalous difference density of a nNOS crystal bound with 3n at 5 σ contour level calculated at (A) the Zn absorption edge (9667 eV, 1.28 Å) and (B) 67 eV away from the edge on the low energy side (9600 eV, 1.29 Å). The Zn peaks are disappeared in panel B because of the weak anomalous signal of Zn at the x-ray wavelength of 1.29 Å, but the anomalous signals from the two heme iron atoms are retained.

Figure 3

The nNOS active site with one molecule of 3j bound above the heme and the other in the pterin binding pocket. The sigmaA-weighted Fo-Fc omit density map for 3j is shown at a 3.0 σ contour level. The ligation bonds around the new Zn²⁺ site and hydrogen bonds are depicted with dashed lines. Two alternate side chain conformations are shown for residue Tyr706. NOS dimerizes through the heme domains with the pterin binding in a pocket at the dimer interface. Residues in subunit A are depicted with green bonds and those of subunit B with cyan bonds. Four pyrrole rings of heme are labeled.

Figure 4

The nNOS active site with one molecule of 3l bound above the heme. The sigmaA-weighted Fo-Fc omit density map for 3l is shown at a 3.0 σ contour level. Major hydrogen bonds are depicted with dashed lines. The second aminopyridine is partially disordered.

Figure 5

The nNOS active site with compound 3n bound. The Fo-Fc omit electron density for the inhibitor is contoured at 3.0 σ. The ligation bonds of the new Zn²⁺ site and hydrogen bonds are depicted with dashed lines. The heme and H₄B in the substrate bound structure (PDB code 1OM4) are overlaid to illustrate the large conformational change of heme propionate A and the displacement of H₄B by the second aminopyridine of inhibitor.

Figure 6

The eNOS active site with compound 3j (A) and 3n (B) bound. The Fo-Fc omit electron density for the inhibitor is contoured at 2.5 σ. Hydrogen bonds are depicted with dashed lines. In panel A, Tyr477 moves to allow interaction of 3j with the heme propionate. In panel B, the second aminopyridine of 3n is partially disordered. The H₄B is not disturbed by ligand binding illustrated by the omit electron density for the pterin at 2.5 σ.

Figure 7

The active site of (A) D597N nNOS or (B) D597N/M336V nNOS mutant with compound 3j bound. The Fo-Fc omit density map for the inhibitor is shown at a 2.5 σ (A) or 3.0 σ (B) contour level. The new Zn site ligation bonds and hydrogen bonds are drawn with dashed lines. Heme propionate A in each case has alternate conformations. In panel A, the second molecule of 3j only partially occupies the pterin site, while H₄B together with two water molecules, Wat1 and Wat2, account for the rest. In panel B, there is no second 3j molecule; hence, H₄B binds with full occupancy indicated by the Fo-Fc omit density map at 3.0 σ.