The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases

Abstract

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity.

Figure 1

Two different modes of binding of 1 to nNOS depending on the chirality at positions 3′ and 4′ of the pyrrolidine. (A) (3R,4R)-1 (PDB entry 3NLM(17)) with its aminopyridine H-bonded with heme propionate D while Tyr706 is in an out-rotamer position. (B) (3S,4S)-1 (PDB entry 3NLK(17)) with its aminopyridine H-bonded with Glu592 while Tyr706 is in an in-rotamer position. All figures were prepared with PyMol (http://www.pymol.org).

Figure 2

NNOS active site bound with (A) (3R,4R)-2 (PDB entry 3UFU(20)) and (B) (3R,4R)-3 (PDB entry 3UFV(20)). The omit F_o – F_c density map for each inhibitor is shown at the 3.0σ contour level. Major hydrogen bonds are depicted as dashed lines.

Figure 3

ENOS active site bound with (A) (3R,4R)-2 and (B) (3R,4R)-3. The omit F_o – F_c density map for each inhibitor is shown at the 3.0σ contour level. Major hydrogen bonds are depicted as dashed lines.

Figure 4

eNOS Y447A mutant active site bound with (A) (3R,4R)-3 and (B) (2R,4S)-6. The omit F_o – F_c density map for each inhibitor is shown at the 3.0σ contour level. Major hydrogen bonds are depicted as dashed lines.

Figure 5

Active site of the nNOS M336V/D597N mutant bound with (A) (3R,4R)-2 and (B) (3R,4R)-3. The aminopyridine and pyrrolidine moieties in (3R,4R)-3 adopt two conformations (yellow and pink) in nNOS. The Tyr706 side chain also has two conformations to accommodate the inhibitor. The omit F_o – F_c density map is contoured at 3.0σ for each inhibitor. Major hydrogen bonds are depicted as dashed lines.

Figure 6

Superimposition of the nNOS–3 structure (purple, shown as one subunit only) on the eNOS–3 structure (green) locally around the heme active site (Pro565–Asp600 in nNOS vs Pro336–Asp371 in eNOS). In nNOS, the loop after Pro338 is invisible, while in eNOS, the loop is ordered up to Leu111, as indicated by the 2F_o – F_c density contoured at 0.8σ. Different binding conformations of (3R,4R)-3 are shown with the Tyr residue swung out (nNOS, purple) or not (eNOS, green). The potential steric clash between Leu111 and Tyr477 in its assumed out-rotamer position is indicated by an arrow.

Figure 7

Segment adjacent to the Tyr706-residing fragment. The normally disordered polypeptide (residues 339–349) becomes partially ordered in certain nNOS structures (PDB entry 4JSH). The 2F_o – F_c electron density is contoured at 0.5σ around the 3₁₀ helix in the segment.

Figure 8

Active site of human iNOS with 7 bound. The omit F_o – F_c density map is contoured at 3.0σ for the inhibitor. Major hydrogen bonds are depicted as dashed lines. The second aminopyridine of 7 is far from heme propionate D, which itself makes an H-bond with Tyr491 in an in-rotamer position.