Oral 1832-2 – Charge Pairing and Phosphorylation Regulate The Conformational Equilibrium and Switching Rates in Neuronal and Endothelial NO Synthase Flavoprotein Domains

Electron flux through nitric oxide synthase reductase (NOSr) is thought to depend on conformational switching motions of their FMN domains, which enables the enzymes to cycle between closed unreactive and open reactive conformational states. However, the conformational equilibrium setpoints (Keq), rates of conformational switching, and interflavin electron transfer rates are mostly unknown, and how these parameters may combine to determine catalytic activities in NOSs is not well understood. To address these, we determined and compared the conformational equilibrium setpoints and rates of conformational switching between reactive open and unreactive closed states, in wild-type nNOSr and four FMN surface mutants (E762R, E762N, E816R, E819R) of nNOSr, and in wild-type eNOSr and the phospho-mimetic S1179D eNOSr mutant. We used stopped flow spectroscopy, single turnover methods, and a kinetic model that relates conformational setpoint and rates of conformational switching to the electron flux through each enzyme to cytochrome c. We found that charge neutralization or reversal at each of these residues alters the setpoint (Keq) of the NOSr conformational equilibrium to favor the open reactive (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the nNOSr mutants suggest that they have higher conformational transition rates (1.5–4-fold) relative to wild-type nNOSr. Wild-type eNOSr mostly exists in closed conformational state (88% closed, 12% open, Keq = 0.125) with a very slow electron flux. In comparison, the S1179D mutation alters the eNOSr setpoint to Keq = 1.5 (40% closed, 60% open), indicating that the open reactive conformation is favored in S1179D eNOSr. Our computer simulation data suggest that S1179D eNOSr also has a faster conformational transition, and a 20-fold faster opening rate relative to wild-type eNOSr. Thus, mutating Ser1179 to Asp alters both the setpoint and transition rates of equilibrium, and these can fully explain the increased electron flux seen in S1179D eNOSr mutant. Together, our studies provide the first measures of conformational equilibrium settings and conformational switching rates in nNOSr and eNOSr proteins, reveal that remarkable differences exist between the two proteins, and show how charge pairing interactions at the domain interface, or phosphorylation at Ser1179, alter NOS activity by modifying these conformational parameters

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18

Effect of NaCl on the thermal deactivation prevention ability of <i>M</i>. <i>leprae</i> HSP18.

<p>The MDH enzyme activity was measured in the absence and presence of 30 μM HSP18 pre-incubated in the absence or presence of 0.05–0.5 M NaCl while it was thermally denatured at 43°C. Similar assays were also performed with 30 μM BSA alone or pre-incubated with 0.05–0.5 M NaCl. Data are means ± standard deviation from triplicate determinations. *<i>p</i>< 0.05, **<i>p</i>< 0.005.</p

Effect of temperature on the structure of <i>M</i>. <i>leprae</i> HSP18.

<p>Far-UV CD spectra <b>(A)</b> and near-UV spectra <b>(B)</b> of HSP18 at different temperatures (25, 31, 37 and 43°C). The concentrations of the protein samples used in far- and near-UV CD experiments were 0.2 and 0.5 mg/ml, respectively. <b>(C)</b> Tryptophan fluorescence spectra of HSP18 (0.05 mg/ml) were recorded from 310–400 nm at various temperatures (25, 31, 37 and 43°C). An excitation wavelength of 295 nm was used. Both the slit widths for excitation and emission were 5 nm. Data were collected at 0.5 nm wavelength resolution. <b>(D)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 were recorded at various temperatures (25, 31, 37 and 43°C). Each of these spectra is an average of 48 scans. For each experiment, spectra were recorded after incubating HSP18 at respective temperature for 1 hr.</p

Secondary structural elements of HSP18 at various temperatures.

<p>Secondary structural elements of HSP18 at various temperatures.</p

Subunit exchange rate constant of <i>M</i>. <i>leprae</i> HSP18 in the absence or presence of 0.05–0.5 M NaCl at 37°C.

<p>Subunit exchange rate constant of <i>M</i>. <i>leprae</i> HSP18 in the absence or presence of 0.05–0.5 M NaCl at 37°C.</p

Determination of subunit exchange rate constant of HSP18 subunits at different temperatures.

<p><b>(A)</b> Time-course alterations in the emission spectrum of Alexa fluor-350 and 488 labeled <i>M</i>. <i>leprae</i> HSP18 due to subunit exchange at 37°C. The emission spectra were recorded at different time points after mixing equal amount of Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 (1 mg/ml each) at 37°C. The fluorescence spectra were recorded from 400 to 600 nm at 37°C using the excitation wavelength of 346 nm. The slit width of both excitation and emission monochromators was 5 nm each. The scan rate used for this assay was 240 nm/min. <b>(B)</b> Time-dependent decrease in fluorescence intensity at 440 nm for data shown in panel A. <b>(C)</b> Time-dependent increase in fluorescence intensity at 513 nm for data shown in panel A. <b>(D)</b> Temperature-dependent subunit exchange rate of <i>M</i>. <i>leprae</i> HSP18. Subunit exchange between Alexa fluor-350 labeled and Alexa fluor-488 labeled HSP18 was monitored at 25, 31, 37 and 43°C. Symbols in panel B, C and D represent the experimental data points and the solid lines in these pannels represent the best fit of the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129734#pone.0129734.e003" target="_blank">Eq 3</a>.</p

Effect of NaCl on the chaperone activity of <i>M</i>. <i>leprae</i> HSP18.

<p>DTT-induced aggregation of 0.35 mg/ml insulin at 25°C <b>(panel A)</b> and thermal aggregation of 0.06 mg/ml CS at 43°C <b>(panel C)</b> in the absence or presence of different HSP18 samples. Both insulin and citrate synthase are denoted as client proteins. Trace 1: Client protein (CP) alone; Trace 2: CP +HSP18; Trace 3: CP +0.05 M NaCl; Trace 4: CP + HSP18 + 0.05 M NaCl; Trace 5: CP +0.15 M NaCl; Trace 6: CP + HSP18+0.15 M NaCl; Trace 7: CP +0.5 M NaCl; Trace 8: CP + HSP18+0.5 M NaCl. Each data point is the average of triplicate measurements. The percent protection ability of different HSP18 samples against insulin and CS aggregation are presented in panels B and D, respectively. The insulin: HSP18 ratio was 1:1.2 (w/w) and the CS: HSP18 ratio was 1:1.5 (w/w). Data are means ± standard deviation from triplicate determinations. NS = Not significant, *<i>p</i>< 0.05, **<i>p</i>< 0.005 and ***<i>p</i>< 0.0005.</p

A proposed model which reflects the schematic mechanism for the chaperone function of <i>M</i>. <i>leprae</i> HSP18 at various temperatures.

<p>A proposed model which reflects the schematic mechanism for the chaperone function of <i>M</i>. <i>leprae</i> HSP18 at various temperatures.</p

Effect of NaCl on the oligomeric mass/size of <i>M</i>. <i>leprae</i> HSP18.

<p><b>(A)</b> Gel filtration profile of HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. TSK-GEL G4000SW_XL column (7.8 mm x 30 cm; 5 μm) was first equilibrated with 50 mM phosphate buffer (pH 7.5) with or without 0.05–0.5 M NaCl. Subsequently, 50 μl of HSP18, pre-incubated without or with 0.05–0.5 M NaCl was injected into the column. The flow rate used in this experiment was 0.5 ml/min. The oligomeric mass of different HSP18 samples was estimated using the standard curve (inset). <b>(B)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 in the absence or presence of 0.05–0.5 M NaCl at 25°C. <b>(C)</b> Intensity particle size distribution spectra of <i>M</i>. <i>leprae</i> HSP18 in the presence of 0.5 M NaCl at different temperatures (25, 31, 37 and 43°C). Each spectrum is an average of 48 scans. HSP18 was incubated at respective temperatures in the absence or presence of 0.05–0.5 M NaCl for 1 hr prior to reading. Protein concentration used was 0.5 mg/ml in 50 mM phosphate buffer, pH 7.5.</p