A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

Abstract

Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg 752 in the FMN subdomain and Glu 47 in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg 752 -Glu 47 bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis. Nitric oxide (NO)4 is an essential signal and effector molecule in biology (1). NO is produced in animals from L-Arg by the NO synthases (NOS, EC 1.14.13.39) (2). Three types of NOS are expressed in mammals: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (3-5). The three NOS are structurally homologous and are active as homodimers (6, 7). Each NOS monomer is comprised of two domains: an N-terminal oxygenase domain (NOSoxy) that contains cofactors protoporphyrin IX (heme) and (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B) and binds the substrate L-Arg, and a C-terminal reductase domain that contains FAD, FMN, and binds NADPH. The NOS reductase domain is homologous to cytochrome P-450 reductase and related dual-flavin enzymes (5, 8), but also contains up to three regulatory inserts that are unique to the NOS enzymes (3, 5). Significantly, a calmodulin (CaM) binding site is located in the connecting sequence between the NOSoxy and reductase domains (3-5). CaM binding to this site activates NO synthesis by triggering electron transfer to the heme in NOS enzymes (9). The ability of CaM to activate a redox enzyme like NOS is novel and the mechanism is a topic of current interest.Much of the actions of CaM impinge on the NOS FMN subdomain, which is thought to undergo large conformational motions to transfer electrons during catalysis (10 -17). This may be a common feature in the dual-flavin reductase enzyme family (18 -20). Fig. 1 illustrates a model for FMN conformational switching during electron transfer between the FMN and heme within a NOS homodimer. The FMN subdomain must first interact with the NADPH-FAD subdomain (FNR) in a "FMN-shielded" conformation to receive electrons, according to equilibrium A. Once the FMN hydroquinone forms (FMNH 2 ), it must swing away to a "FMN-deshielded" conformation, and then must interact with the NOSoxy do...

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

“…From our model the FMN domain does not directly contact the oxygenase domain, as proposed from previous work (21,25). However, a minor rotation of the connecting arm would bring the domains closer and position the FMN adjacent the heme pocket.…”

“…A conserved reductase domain Arg residue (Arg-753 in nNOS), identified to form a salt bridge with CaM in the iNOS FMN domain-CaM structure, was shown in recent studies to be important for enhancing CaM-dependent catalytic activity (19,21,44). Our results provide additional structural framework for this interaction and support a model whereby this salt bridge and other interactions that stabilize the FMN domain-CaM arrangement function to drive the release of the FMN domain from the shielded state and stabilize the open-clamp conformation we have identified.…”

“…CaM interactions increase FMNheme electron transfer, possibly by destabilizing the shielded state through bridging contacts with the FMN or interactions with the reductase connecting domain (CD) (16 -20). Electrostatic interactions via structural control elements in the reductase domain, including the autoinhibitory loop, C-terminal tail, and connecting domain, favor the shielded state and are proposed to become disrupted for precise release of the FMN domain during the catalytic cycle (21,22).…”

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

“…This slow rate of electron transfer from FMN to heme is due to the conformational change of the FMN subdomain required to shuttle electrons between the input and output states (3). This large conformational change is articulated at the hinges surrounding the calmodulin-binding helix (9,33) (Fig. 7A).…”

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

“…Sample Preparation and Synchrotron X-ray Radiolysis-Recombinant bovine calmodulin (CaM) was purified as described previously (28). The CaM protein sample was diluted into 10 mM cacodylic acid sodium salt trihydrate, 0.2 mM CaCl 2 , pH 7.5, to the final concentration of 6 M. Radiolysis experiments were performed at the beamline X-28C of the National Synchrotron Light Source with the ring energy of 2.8 giga electron volts and beam current ranging between 190 -210 mA (29 -31).…”