N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization

Abstract

Nitric oxide synthase oxygenase domains (NOS ox ) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS ox reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal β-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra-to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS ox molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

“…Zn 2+ was reported to be required for the stability of NOS, but not for the catalytic reaction itself [30][31][32]. In contrast with NOS, our data demonstrate that Zn 2+ is essential for the activity of the GCH enzyme.…”

Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans

“…NOS sequences are colorcoded to highlight zinc ligands Cys-104 and Cys-109 and proximal heme ligand Cys-194 (yellow background), Arg-binding residues (cyan letters), H 4B-binding residues (red letters), and residues that form the active-center channel leading to the heme (cyan boxed). Dimer interface residues that contribute at least 5 Å 2 of buried surface area in the unswapped state of the inducible NOSoxy domain (34) are shown with green background. Above, black arrows show ␤-strands, white boxes show ␣-helices.…”

“…Seven additional C-terminal deiNOS residues (TGHAPTG) do not have analogs in the NOSoxy structures and are thus not shown. (B) Ribbon representation of the unswapped iNOSoxy dimer (34) highlighting the structural elements absent in deiNOS and surface residues conserved among NOSs. Most of the NOSoxy core (purple and red subunits) is conserved by deiNOS with the exception of the N-terminal hook, switch region, N-terminal binding pterin segments, and two peripheral loops (cyan and orange).…”

“…Notably, the intermolecular zinc binding site (gray, bottom center) is not found in deiNOS, although regions that bind heme (yellow bonds) and H 4B (yellow, center, on edge) are conserved. Residues that form a conserved surface patch above the active center channel and surrounding an exposed heme edge in mammalian NOSoxys (34) and are also conserved in deiNOS are highlighted (green side chains) and numbered. that participate in the dimer interface or substrate binding channel are somewhat less conserved between deiNOS and iNOS.…”

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

“…The NOSox domain is built from a unique, winged β-sheet fold (Crane et al, 1997). NOSox binds L-arginine substrate, heme, and two tetrahydrobiopterin cofactors and a zinc ion that stabilize the NOS dimer interface (Crane et al, 1998,Raman et al, 1998,Crane et al, 1999,Fischmann et al, 1999. NOSox accepts electrons from NOSred to catalyze the sequential monooxygenation of L-arginine, into Nhydroxyarginine and then into citrulline and NO.…”