title: Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase.
abstract: The nitric oxide synthase (NOS) dimer is stabilized by a Zn(2+) 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 Zn(2+). 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 Zn(2+) 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 Zn(2+) binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn(2+) binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn(2+) site. Addition of excess Zn(2+) stabilizes the Zn(2+) 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 Zn(2+) site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn(2+), pterin, and substrate binding sites.
authors.affiliation: Departments of †Molecular Biology and Biochemistry, ‡Chemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697-3900, United States.
authors.forename: Georges
authors.initials: G
authors.lastname: Chreifi
authors.forename: Huiying
authors.initials: H
authors.lastname: Li
authors.forename: Craig R
authors.initials: CR
authors.lastname: McInnes
authors.forename: Colin L
authors.initials: CL
authors.lastname: Gibson
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Suckling
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D002417:Cattle; D018360:Crystallography, X-Ray; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization; D015032:Zinc / Q000737:chemistry*
chemical_list: D001708:Biopterins; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D015032:Zinc