Positional Distributions of the Tethered Modules in Nitric Oxide Synthase: Monte Carlo Calculations and Pulsed EPR Measurements

Abstract

The nitric oxide synthase (NOS) enzyme consists of multiple domains connected by flexible random coil tethers. In a catalytic cycle, the NOS domains move within the limits determined by the length and flexibility of the interdomain tethers and form docking complexes with each other. This process represents a key component of the electron transport from the flavin adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate binding domain to the catalytic heme centers located in the oxygenase domain. Studying the conformational behavior of NOS is therefore imperative for a full understanding of the overall catalytic mechanism. In this work, we have investigated the equilibrium positional distributions of the NOS domains and the bound calmodulin (CaM) by using Monte Carlo calculations of the NOS conformations. As a main experimental reference, we have used the magnetic dipole interaction between a bifunctional spin label attached to T34C/S38C mutant CaM and the NOS heme centers, which was measured by pulsed electron paramagnetic resonance. In general, the calculations of the conformational distributions allow one to determine the range and statistics of positions occupied by the tethered protein domains, assess the crowding effect of the multiple domains on each other, evaluate the accessibility of various potential domain docking sites, and estimate the interaction energies required to achieve target populations of the docked states. In the particular application described here, we have established the specific mechanisms by which the bound CaM facilitates the flavin mononucleotide (FMN)/heme interdomain docking in NOS. We have also shown that the intersubunit FMN/heme domain docking and electron transfer in the homodimeric NOS protein are dictated by the existing structural makeup of the protein. Finally, from comparison of the calculated and experimental docking probabilities, the characteristic stabilization energies for the CaM/heme domain and the FMN domain/heme domain docking complexes have been estimated as -4.5kT and -10.5kT, respectively.

Fig. 1.

The structural model used for calculating the conformational distribution of rat nNOS. The heme domain was represented by a crystal structure (pdb 4JSH), while all other structural elements (the amino acid residues of the tethers, CaM, and the FMN and FNR domains) were modeled by spheres with the radii indicated in the Figure. The amino acid sequence numbers of the tethers are indicated in red.

Fig. 2.

A chain fragment showing the definition of the propagation angles θ and φ.

Fig. 3.

The distance dependence of the interaction energy described by Eq. 2.

Fig. 4.

Calculated positional distributions of the centers of FNR domain (a, d), FMN domain (b, e), and bound CaM (c, f) in full-length nNOS. Panels a - c show the pseudouniform distributions calculated for all E_d values equal to zero. The distributions in panels d – f are calculated for E_d(CaM) = −4.5kT, E_d(FMN) = −10.5kT, E_d(FF) = 0, and R_o = 5 Å. The area around the distribution maxima is highlighted by the black color. The heme domain is shown in cyan. Brown circles indicate the positions of the heme centers. Blue circle shows the position of the tether origin. The yellow circle with a black border shows the predicted docking position of the module shown in the given panel.

Fig. 5.

P_d(CaM) and P_d(FMN) dependences on E_d(CaM) and E_d(FMN), respectively, calculated for nNOS oxyFMN construct using the Monte Carlo approach (circles) and Eq. 3 (dashed lines). Red circles, P_d(FMN) in the absence of CaM; blue filled circles, P_d(FMN) in the presence of bound CaM, but with E_d(CaM) = 0; blue open circles, P_d(FMN) in the presence of bound CaM, with E_d(CaM) = −12.5kT (corresponds to P_d(CaM) ~ 1); black filled circles, P_d(CaM) for E_d(FMN) = 0; black open circles, P_d(CaM) for E_d(FMN) = −17.5kT (corresponds to P_d(FMN) ~ 1). Dashed lines approximating the dependences given by circles are calculated using Eq. 3 with E_u = 0 and V_u/V_d = 160000 (for red circles), 105000 (blue filled circles), 19000 (blue open circles), 600 (black filled circles), and 270 (black open circles).

Fig. 6.

RIDME traces recorded for BSL CaM at T_low = 8 K (panel a) and T_high = 20 K (panel b). Traces 1, 2, and 3 in each panel correspond to nNOS oxyFMN with bound CaM, full-length nNOS with bound CaM, and full-length nNOS with unbound CaM, respectively. Experimental conditions: mw frequency, 34.612 GHz; mw pulses, 10, 10, 10, and 14 ns; T_R = 65 μs; magnetic field, B_o = 1.2325 T (corresponds to the maximum of the SL field sweep spectrum), pulse repetition rate, 5 Hz.

Fig. 7.

Comparison of the experimental and calculated quotient RIDME traces for nNOS oxyFMN. Black line, the quotient RIDME trace obtained from the experimental nNOS oxyFMN data (traces 1 in Fig. 6) using Eq. 5. Blue line, RIDME trace calculated based on the pseudouniform conformational distribution. Red line, RIDME trace calculated based on the conformational distribution obtained using R_o = 5 Å, E_d(CaM) = −4.5kT, and E_d(FMN) = −10.5kT.

Fig. 8.

Results of calculations of nNOS conformations, which give reasonable fits of the quotient RIDME trace shown in Fig. 7. Panel a shows the pairs of E_d(FMN) and E_d(CaM) values; panels b and c show the corresponding P_d(CaM) and P_d(FMN) values, respectively. Black, red, and blue circles and lines correspond to R_o = 0 Å, 5 Å, and 10 Å, respectively. Horizontal yellow regions show the ranges of P_d(CaM) and P_d(FMN) values, which correspond to the (expected) experimental values. The gray vertical region corresponds to the range of situations when P_d(CaM) and P_d(FMN) calculated for R_o = 5 Å simultaneously agree with the experimental values.

Fig. 9.

Comparison of the experimental and calculated quotient RIDME traces for the full-length nNOS enzyme. Black line, the quotient RIDME trace obtained from the experimental data (traces 2 in Fig. 6) using Eq. 5. Gray line, the quotient RIDME trace of the oxyFMN construct reproduced from Fig. 7. Blue line, RIDME trace calculated based on the conformational distribution obtained using E_d(FF) = 0. Red line, RIDME trace calculated based on the conformational distribution obtained with E_d(FF) = −10.5kT. Other calculation parameters were the same as in the calculation for the oxyFMN construct: R_o = 5 Å, E_d(CaM) = −4.5kT, and E_d(FMN) = −10.5kT.