Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study

Abstract

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production.

Keywords: Calmodulin; Electron transfer; Heme; Molecular dynamics; Nitric oxide synthase.

Figure 1

The FMN domain tethered shuttle model (with the tethers corresponding to the interdomain FMN-heme and FAD-FMN connectors). The FMN domain (cyan) shuttles between the NADPH-FAD domain (white) and the heme-containing oxygenase domain (red). CaM (green) binding to eNOS/nNOS unlocks the NADPH-FAD/FMN domain interacting state (i.e., input state), thereby enabling the FMN domain to shuttle between the FAD and heme domains. The input state and FMN/heme domain interacting state (i.e., output state) are well defined, while free FMN domain conformations also exist in between these two docked states. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 2

Initial docked structure of the FMN domain (pink) and the dimeric heme domains A & B (green and red) along with CaM-binding motif (yellow) bound with CaM (cyan). The missing linker residues (in black) were added by using Scigress and further refined by energy minimization using NAMD software. Note that the FMN domain in subunit B docks to the heme domain in subunit A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 3

Root-mean-square deviation (rmsd) for the backbone of human iNOS oxyFMN•CaM at the pre-IET [Fe(III)][FMNH^–] state (a, b) and the post-IET [Fe(II)][FMNH^•] state (c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 4

The Fe···N₅(FMN) distance in the pre- (red, green) and post-IET (blue) states during the MD simulations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 5

Typical IET-competent NOS conformations with the FMN and heme molecules shown in green and yellow Licorice mode, respectively. In the three conformations the FMN approaches different sides of the heme porphyrin ring. (a) Conformation 1: IET presumably takes place through from FMN → Phe593 → Trp372 → heme center. (c) Conformation 2: IET takes place through FMN → Trp372 → heme center. (e) Conformation 3: IET takes place through FMN → Tyr631 → Trp372 → heme center. (b, d, f) The B3LYP computed molecular electrostatic potentials for the conformations 1, 2, 3, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 6

Selected key residues in the interfaces between heme, FMN and CaM domains in the pre-IET state. Heme A domain, heme B domain and CaM are represented as blue, pink, and cyan ribbon, respectively. The FMN domain is shown as yellow ribbon. Refer to Figures 7-8 for the detail information on the residue-residue interactions on each interface. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 7

(a) During the 60 ns simulation course of the pre-IET state, the distances between heme A domain residues Arg86 guanidinium carbon atom (CZ) and Glu479 carboxylate group carbon (CD) atom (red trajectory), between heme A domain residue Arg86 CZ atom and CaM residue Asp122 carboxylate carbon (CG) atom (green trajectory), and between heme A residue Arg83 CZ atom and CaM residue Asp122 CG atom (blue trajectory). (b) Initially, heme A domain residue Arg86 (green Licorice mode) forms a salt bridge with Glu479 (red Licorice mode) of the same domain. (c) At ~ 22 ns, the Arg86 residue (green Licorice mode) forms a salt bridge with CaM residue Asp122 (purple Licorice mode). (d) At ~ 40 ns, heme A domain residue Arg83 (yellow Licorice mode) forms a salt bridge with CaM residue Asp122 (purple Licorice mode). Heme A domain, heme B domain and CaM are represented as blue, pink, and cyan ribbon, respectively. The FMN domain is shown as yellow ribbon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 8

Salt bridges between CaM (cyan) and the linker region of the heme B domain (red) in the pre-IET state. The negatively charged CaM residues (Glu14, Glu120, Glu123, Glu127) are labeled in red. The positively charged linker residues Lys505, Arg506, Arg510, and Lys509 are labeled in blue. The FMN domain is in yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 9

Inter-domain interactions in the post-IET state. Heme A, heme B, CaM and FMN domains are represented in pale blue, red, pink and violet ribbon, respectively. (a) The Fe···N₅(FMN) distance at this snapshot amounts to 27.4 Å. (b) The interactions between the heme A and CaM domains. (c) The interactions between the heme B and CaM domains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure 10

(a) The heme domains in two conformations at 60 ns of human iNOS oxyFMN•CaM in the pre- and post-IET states are superimposed to illustrate the motions of the FMN domain (colored in yellow and pink for the pre- and post-IET states, respectively) and CaM (pre-IET: cyan; post-IET: purple) (b) CaM in the post-IET state (violet) and the pre-IET state (cyan: N-terminal, green: C-terminal, mauve: central linker); the CaM-binding helix of NOS in the pre- and post-IET states is colored yellow and bazaar, respectively. Calcium ions are shown in spheres and labeled.

Figure 11

Force profile from the SMD simulation when the FMN domain is further pulled away from heme A domain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)