Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

Abstract

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2؉ -calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20 -40-amino acid tail that attenuates electron transfer in a CaMindependent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834 KGSPG-GPPPG 843 , and is truncated to 809 ESGSY 813 (iCD2A) in iNOS. The eCD2A contributes to the Ca 2؉ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.Nitric-oxide synthase (NOS), 1 a modular protein, consists of an N-terminal heme domain that catalyzes P450-like oxidations and a C-terminal two-flavin domain homologous to that of cytochrome P450 reductase (CPR) (1, 2). The NOS oxygenase and reductase domains are linked by a calmodulin (CaM) binding helix. The NOS oxygenase domain is completely different from a P450, however, in that it has no sequence identity with that family of enzymes and has an ␣,␤-fold structure distinct from the highly ␣-helical structure of the P450 enzymes (3-6). Furthermore, the two-stage mechanism for the oxidation of L-arginine to L-citrulline and nitric oxide (NO) by NOS is more complex than that of a P450 enzyme, as it requires tetrahydrobiopterin (H 4 B) as a transient electron donor (7). In contrast, the NOS reductase domain exhibits considerable sequence identity with CPR, and the electrons, as in P450/CPR, flow from NADPH to the FAD, then to the FMN, and finally, upon substrate binding, to the heme (2, 8).In the cytochrome P450 system, the principal control on inter-protein electron transfer is the requirement for association of the P450 with CPR. The NOS complex also requires interactions between independently transcribed proteins because it is a homodimer in which the reductase domain of one subunit provides electrons to the oxygenase domain of the other (9, 10). However, inter-domain electron transfer in NOS also requires the binding of CaM, and the Ca 2ϩ...

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

“…This 15-residue ␤-finger structure, called the SI for "small insertion," is adjacent to the edge of the FMN-binding subdomain, proximate to the AR and, by inference, the CaM-binding site (52). This element, like AR, is inhibitory for electron transfer and NO production (52,53) in the absence of CaM. Its function appears to be masked in the presence of the AR, and it is only when AR is deleted that the regulatory effects of SI become apparent (53).…”

Endothelial Nitric-oxide Synthase Activation Generates an Inducible Nitric-oxide Synthase-like Output of Nitric Oxide in Inflamed Endothelium

“…Although the mechanism of this enhanced Ca 2ϩ -CaM binding to eNOS is unknown, it could involve altering the phosphorylation state of residues in or near the Ca 2ϩ -CaM binding region. Three domains in eNOS inhibit its interaction with CaM at resting Ca 2ϩ levels as follows: 1) an auto-inhibitory loop (amino acids 595-639) located within the FMN binding domain (63,64); 2) a C-terminal tail extension (amino acids 1165-1178) present in the reductase domain (65); and 3) a highly conserved loop (CD2A) within the connecting domain (66). Phosphorylation at several sites on eNOS can increase its sensitivity to [Ca 2ϩ ] i most likely through disruption of one or more of the control elements (e.g.…”

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

“…In addition, eNOS and nNOS contain the 42-45-residue auto-inhibitory helix (AH) within the FMN-binding domain, which interferes with Ca 2+ /CaM binding and inhibits both intra-and inter-module electron transfers. Also, a regulatory element, the 'CD2A' loop of a protruding β-finger present in the CD of eNOS and nNOS, plays an autoinhibitory role in the control of NO by interaction with the CaM-binding peptide (Knudsen et al, 2003). The upregulation of eNOS and nNOS activity is controlled by phosphorylation of both the CT and AH regulatory elements and by reversible Ca 2+ /CaM-binding.…”