Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism
© Wei et al 2011
Received: 8 February 2012
Accepted: 10 April 2012
Published: 10 April 2012
Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.
The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD.
We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific.
KeywordsEF-hand Calcium binding NADPH Oxidase 5 Fluorescence Isothermal titration calorimetry
Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, play important roles in host defense, signal transduction, and hormone synthesis . Superoxide is primarily generated by the enzyme family of NADPH oxidases (NOXs), and several homologous enzymes have been identified, including NOX1, NOX2, NOX3, NOX4, NOX5, and dual oxidase (DUOX) [2, 3]. Among them, NOX2 (a.k.a. phagocyte NOX) serves primarily in nonspecific immunity by generating superoxide which kills invading microbes . While a large amount of superoxide is essential in phagocytic cells, trace amounts of superoxide generated from other NOX enzymes are required for non-phagocytic cells to function properly. Accordingly, deficiency or overproduction of superoxide has been demonstrated to lead to pathological problems, as well as neurological, cardiovascular, and renal diseases.
Using flow dialysis, Banfi et al. determined the macroscopic (stoichiometric) binding constants and derived their intrinsic binding constants (Ka) for the binding of the 1st - 4th Ca2+ ions to CaBD assuming independent binding among Ca2+ binding sites . They concluded that the N-terminal half (NCaBD), containing the 1st and 2nd EF-hand (EF1 and EF2) motifs, has a lower calcium binding affinity, with Ka values ranging from 1.7 × 104 to 6.8 × 104 M-1, while the C-terminal half (CCaBD), containing the 3rd and 4th EF-hand motifs, has a high calcium binding with Ka values ranging from 1.6 × 105 to 9.5 × 105 M-1. They then demonstrated that the two halves functions independently in terms of Ca2+ binding affinities. Previously, we have shown that the higher Ca2+ affinity of the C-terminal half might be attributed to its slower Ca2+ dissociation using full-length CaBD and a mutant (CaBD(E99Q/E143Q)) in which the Ca2+ binding of EF3 and EF4 is impaired . Further, our data suggest that the two halves have different metal binding properties. To ambiguously confirm our previous conclusion about the N-terminal half of CaBD, which require excluding the contribution of Trp fluorescence and secondary structural change from the C-terminal half, we specifically generated the recombinant N-terminal half of CaBD and its mutants, and separately studied their metal binding using fluorescence, isothermal titration calorimetry, and circular dichroism. Our results supported our previous findings about the different metal binding characteristics and revealed a possible mechanism for Ca2+ binding to the 1st and 2nd EF-hands of NOX5. The binding thermodynamic parameter, such as enthalpy difference (ΔH), and structural change for Ca2+ binding are also presented.
Results and discussion
Effects of metal ions on trp fluorescence of NCaBD and its mutants
To better understand what role the individual EF-hands in NCaBD play in metal binding, we generated three mutants, NCaBD(E31Q), NCaBD(E63Q) and NCaBD(E31Q/E63Q), in which the Glu residues at the -Z (+12) position of the corresponding EF-hands were replaced with Gln. Such mutation approach has been employed to impair calcium binding affinity in NOX5 [6, 7] and other Ca2+ binding proteins . In order to better associate Ca2+ binding with the individual EF-hands, we refer to NCaBD(E31Q) as -1EF+2EF to emphasize that its Ca2+ binding site at the 1st EF-hand is "removed" while the 2nd EF-hand is intact. Similarly, NCaBD(E63Q) and NCaBD(E31Q/E63Q) are referred as +1EF-2EF and -1EF-2EF, respectively. The fluorescence of -1EF+2EF enhances ~20% upon Ca2+ binding and 50 μM Ca2+ is enough to saturate the binding (data not shown). The fluorescence of +1EF-2EF was also enhanced ~20% upon Ca2+ binding (upper panel in Figure 2B). Interestingly, its binding is very weak; approximately 4 mM Ca2+ was required to saturate the binding. Since ITC provides more binding characteristics than fluorescence, we used it to determine the Ca2+ binding constants and the corresponding thermodynamic parameters. However the lower Ca2+ affinity of +1EF-2EF allowed us to perform a direct titration using fluorescence since the free Ca2+ concentration is roughly equal to the total Ca2+ concentration for a weak binding. The titration revealed that its Ca2+ binding is best fitted with cooperative binding (nH = 1.50) (lower panel in Figure 2B). Note that the observed binding constant, Kobs, obtained from Hill equation is not related to either macroscopic or microscopic binding constant. We used a model with two binding sites and determine its Ka values to be 6.1 × 102 and 2.2 × 102 M-1 (data not shown). The fluorescence titration indicates that the mutated EF2 still retains its Ca2+-binding ability, albeit much weaker, which is affected by the Ca2+-bound EF1 in a positive cooperative manner. By combining the measurement from ITC (see below), we estimated Ka for EF1 to be ~ 5 × 102 M-1. The double mutant, -1EF-2EF, showed no response to Ca2+ up to 20 mM, indicating that replacing Glu with Gln has completely abolished Ca2+ binding for all EF-hands. Measurements with Ca2+ concentration higher than 20 mM Ca2+ poses difficulties because it decreases fluorescence intensity as protein solubility decreases and ionic quenching occurs. Thus, replacing Glu to Gln at both EF-hands results in very weak Ca2+ binding (< 50 M-1), which is not biologically significant.
Previously, we characterized the AEDANS labeled CaBD and CaBD(E99Q/E143Q), in which the fluorescent dye is labeled at cys-107 that is located in EF3 and EF4. The labeled proteins are believed to be more sensitive to the structural change of the C-terminal half and our data suggest that only the C-terminus half of CaBD binds to Mg2+ . We included up to 20 mM Mg2+ to the wild type and the mutants of the N-terminal half domain but no significant fluorescence change was observed, thus confirming our previous conclusion.
Stern-Volmer Quenching Constants Obtained from Acrylamide Titration
5.28 ± 0.22
5.04 ± 0.35
9.49 ± 0.52
6.95 ± 0.22
6.78 ± 0.35
12.66 ± 0.52
6.61 ± 0.17
6.68 ± 0.33
9.28 ± 0.08
6.70 ± 0.21
6.71 ± 0.15
9.51 ± 0.16
7.40 ± 0.42
7.35 ± 0.35
7.45 ± 0.41
Secondary structures determined by circular dichroism
Isothermal titration calorimetry
The Ca2+ Binding constants for the 1st and 2nd EF-hands
Ka M-1 ( ΔH kJ/mol)
1.7 -6.8 × 104
3.05 ± 1.02 × 105 (-4.96 ± 0.25), 8.60 ± 1.65 × 106 (-24.88 ± 0.43)
-1EF + 2EF
6.51 ± 0.80 × 105 (-14.3 ± 0.3)
~ 5 × 102 (-4.0 ± 0.2)
As expected, the Ca2+ titration of -1EF+2EF measured by ITC can be fit to a model with one binding site and yielded the values of Ka and ΔH to be 6.5 × 105 M-1 and -14.1 kJ/mol, respectively (Figure 4B). The heat released due to Ca2+ binding to EF2 alone is between the ΔH values for the 1st and 2nd Ca2+ binding, suggesting that the model with independent binding is unlikely. The binding affinity of EF2 is comparable to the values derived from the ITC analysis of NCaBD. For +1EF-2EF, the signal to noise ratio (S/N) of the ITC signal was too small to make a precise determination of its binding constant (Figure 4C). The Ka value was estimated to be ~800 M-1 for one-binding site or ~400 M-1 for two identical binding sites, which is consistent with the value determined from fluorescence characterization (Figure 2B). The value of ΔH was calculated to either -8 kJ/mol for a model with one binding site or -4 kJ/mol for a model with two identical binding sites. Total heat generated for Ca2+ binding to +1EF-2EF was further confirmed using a separate continuous titration method that determines ΔH directly (data not shown).
Although Mg2+ binding was not observed in our spectroscopic study, one can not completely rule out the possibility since the Trp fluorescence or CD only reflects the change on Trp residue(s) or the secondary content change, respectively. For example, the C-terminal half of CaBD contains one Trp residue that is distant from its EF-hands and does not change its Trp fluorescence upon Ca2+ binding (ref.  and our unpublished result). Mg2+ binding most likely changes only local structures, enabling no significant signal in CD and fluorescence. Because it is not possible to extract useful binding parameters using a direct ITC titration for a low Mg2+ binding, we performed a competitive ITC binding experiment by titrating Ca2 + to a solution containing the protein plus 20 mM Mg2+. If Mg2+ does bind to our proteins, the additional Ca2+ ions has to compete with the same binding site(s) occupied by Mg2+, resulting in lower binding affinity. We have not observed any ITC binding change.
Previously, we generated a CaBD mutant, (E99Q/E143Q) or +1EF+2EF-3EF-4EF , which presumably functions as NCaBD. To check if this is the case, we thus measured its Ca2+ binding by ITC. We observed an identical binding isotherm as that of NCaBD (data not shown) and the results also indicate that replacing Glu with Gln in EF3 and EF4 does impair their Ca2+ binding significantly.
The process by which Ca2+ induces conformational change in NOX5's CaBD is critical for the inter domain-domain interaction and is required in order to produce superoxide. The interaction appears to be mediated by the four EF-hands it contains. In the past decade, characterization of a variety of calcium binding proteins provides a wealth of information about how Ca2+ binding to their individual EF-hands leads to biological responses. However, it is impossible to predict Ca2+ binding strength and its cooperativity simply based on the sequences of EF-loop because the Ca2+ binding is dependent upon the overall structure not simply the loop change . The characterization of the non-canonical EF1 and canonical EF2 reveals that Ca2+ binding to NCaBD induces a structural change as observed in the enhancement of Trp fluorescence and the increase of α secondary structure. The Trp fluorescence results show that λmax is shifted from 340 in CaBD to 350 nm in NCaBD upon Ca2+ binding, suggesting that the red spectrum shift is caused by Trp residues being more exposed to solvent when the C-terminal half is not present. This explains equally well the higher Ksv values for the apo NCaBD than the apo CaBD and the Ca2+-bound NCaBD than Ca2+-bound CaBD that were observed (Table 1).
Data analysis of Ca2+ binding affinities to NCaBD obtained by ITC titration indicates that the Ca2+ binding to EF1 and EF2 could be explained by a model with two independent binding sites or identical intrinsic binding with a slightly negative cooperative. The data is somewhat parallel to the flow dialysis data that assume no interaction between EF-hands and, thus, the corresponding intrinsic binding constants were derived by statistics. However, analysis using either model provides no information about the Ca2+ binding for each EF-hand. Given a very weak macroscopic cooperative binding as observed in NCaBD, it is fair to assume that NCaBD contains a non-interacting pair of EF-hands, which is in agreement with the assumption made in a previous report using Adair equation . However, our ITC data clearly indicates that the macroscopic and microscopic binding constants for NCaBD, assuming an independent model, are one-order higher than the reported values, and also the binding for one site is one order stronger than the other (Table 2).
While there is no indication that individual EF-hands interact with each other due to the fact that the ITC and fluorescence studies of Ca2+ binding to NCaBD could only provide macroscopic view of Ca2+ binding, mutagenesis studies on individual EF-hands do indicate that this independent binding model is unlikely. The NOX5's 2nd EF-loop has a conserved canonical EF-hand sequence (Figure 1A) and also shows a strong Ca2+ binding (Ka = ~ 106 M-1), suggesting its Ca2+ model is similar to known canonical EF-hands by chelating Ca2+ to the residues at +X (1), +Y (3), +Z (5), -Y (7), and -Z (12) with a water molecule through the residue at -X (9) (Figure 1B). The non-canonical EF1 alone has a very weak Ca2+ binding with a ΔH value of -4 kJ/mol. Because CD and quenching studies indicate there is no structural difference between the wild type and mutants in the absence of Ca2+, the low Ca2+ binding affinity of EF1 could not simply be explained by structural alternation due to mutation. The EF-loop of EF1 contains only 11 amino acids with non-prototypic acid residues in +X and +Y positions, which is found in most members of the penta-EF-hand superfamily such as sorcin, calpain, and grancalcin. Although the binding affinity of non-canonical EF-hands is not necessarily weaker than canonical EF-hands, the low binding affinity of EF1 might be attributed to the decrease in entropic contribution. In caplain domain VI (dVI) the non-canonical EF1 (Figure 1C), which shares similar sequence to NOX5' EF1, adopts an alternative conformation by chelating Ca2+ to the oxygen molecule of a main-chain carbonyl group (+X) and to an additional water molecule (+Y) [12, 13]. Thus with two "frozen" water ligands remaining within EF-loop, the affinity is decreased. Thus our data suggests that in order for NOX5 EF1 to adopt an optimal conformation for Ca2+ binding, the structural change on EF2 upon Ca2+ binding is essential to increase its sensitivity.
It is believed that Mg2+ binding to EF-hands is biologically important. EF-hands bind to Mg2+ in a substantially different way than Ca2+; it causes conformational change locally and is unable to elicit enzymatic activity. In the resting cells, the concentration of Mg2+ is 0.5-2 mM and thus it is a potent competitor for EF-hands. Here, we concluded unambiguously that Mg2+ binding to NCaBD is not biologically important. To our best knowledge, there is no report of Mg2+ binding to non-canonical EF-hands. For canonical EF-hands, an acid-pair theory, which states that the strength of Mg2+ binding is proportional to the numbers of X, Y, Z acid pairs (i.e. the X-acidic pair contains two acidic residues, Asp or Glu, in +X and -X positions), has been used to explain Mg2+ binding affinity . In agreement with this theory, the NOX5's EF2 contains no acidic pairs and shows no Mg2+ binding.
Ca2+ binding to NOX5 was determined to be weak and, because of this, a question arises concerning whether the Ca2+ level (~10-5 M) in activated cells is able to saturate and activate NOX5. Here, we show that NOX5's EF1 and EF2 possess a higher binding affinity than the reported values. Our preliminary results indicate that Ca2+ binding to CaBD gates heme reduction using membrane solublized NOX5 and enhances the electron transfer from NADPH to FAD in the flavoprotein domain, which was determined using cytochrome C assay (unpublished results). We are investigating how the individual EF-hands impact on the NOX5 superoxide-generating activity. Recent studies indicate that NOX5's calcium sensitivity is enhanced when its flavoprotein domain interacts with CaM  or is phosphorylated at its Thr494 and Ser498 residues . All evidence suggests that the elevation of Ca2+ flux is critical for NOX5 activation while other factors further regulate its superoxide-generating activity. Because the activation still relies on Ca2+ binding, exactly how the structural change in the C-terminal flavoprotein is induced by the above mechanisms and propagates to CaBD remains to be studied further. Studies examining Ca2+ binding to individual NOX5's EF-hands are important for further elucidation of the mechanism.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific, and were used without further purification. All buffers used in the protein characterization were rendered "calcium-free" by treatment with Chelex-100 (Bio-Rad Laboratories, Hercules, CA). The measurements were performed at least three times using at least three different protein batches, and the results were reproducible.
The plasmid pLW-His6-NCaBD (residues 1-78) was generated as described previously . All constructs contain an extra 11 residues, MAHHHHHHAVP, at their N-terminal ends. The mutants NCaBD(E31Q), NCaBD(E63Q) and NCaBD(E31Q/E63Q) were created using the PFusion Mutation kit (New England Biolabs, Ipswich, MA). Polymerase chain reactions (PCRs) were performed using an Eppendorf MasterCycler ep thermocycler (Hauppauge, NY). The oligonucleotides for PCR were synthesized by Integrated DNA Technologies (Coralville, IA). The desired mutations in plasmid constructs were confirmed either by DNA sequencing or silent-digestion.
Recombinant protein expression and purification
The protein expression was performed as described previously . In general, T7 E. Coli cells (New England Biolabs) carrying the desired plasmid were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 = 0.6-0.8 and the culture was continuously incubated for an additional 4-6 h at 37°C. Cells were then homogenized and the released His-tagged proteins were purified by chromatography. NCaBD was purified through a phenyl-sepharose column as described for NOX5's CaBD . Mutants were first purified through a Ni-NTA column followed by a DEAE cellulose (Sigma-Aldrich) column (2.5 × 10 cm) using a BioLog FPLC System (Bio-Rad Laboratories). The flow rate was 1 mL/min. The DEAE column was equilibrated with a buffer containing 50 mM Tris, pH 7.5 (Buffer A) and the desired protein was eluted with a linear gradient from 0 to 100% Buffer B (50 mM Tris, pH 7.5, 0.3 M NaCl) over approximately 1.5 h. NCaBD(E31Q), NCaBD(E63Q), NCaBD(E31Q/E63Q) were eluted out at 54-69%, 50-65%, and 47-61% buffer B, respectively. To render the protein Ca2+-free, an EDTA solution was added to the protein samples to make the final concentration of 5 mM followed by dialysis against a 1-L buffer containing 50 mM Tris, pH 7.5 and 1 mM EDTA overnight at 4°C. The proteins were then buffer-exchanged with Chelex-treated buffer in an Amicon Ultra centrifugal filter device (3,000 Da MWCO) or by gel filtration (Sephadex G-25). The purified proteins were stored at -80°C until use. The protein purity was checked on SDS electrophoresis and its purity was estimated > 90% based on density profiles measured using UN-SCAN-IT software (Silk Scientific, Inc, Utah). The SDS-PAGE picture of purified proteins used in this study is shown in Additional file 2: Figure S2. The protein concentration was estimated with the Bradford assay (Bio-Rad Laboratories) using bovine serum albumin (BSA) as standard.
Isothermal titration calorimetry
The ITC experiments were carried out on a Nano-ITC2G instrument (TA instruments, DE) at 25°C. All samples were degassed before loading. The syringe and reaction cell were treated with a solution containing 100 μM EDTA to remove any residual Ca2+ followed by an exhaustive rinse of Chelex-treated water and buffer. A typical titration was performed by sequential injections every 400 s of 1.6 to 4 μL of a 3-5 mM Ca2+ (or 10-30 mM Mg2+) solution into a hastelloy reaction cell (0.94 mL) that contained 0.0375-0.160 mM protein. The sample cell was stirred continuously at 200 rpm. Samples were re-examined for precipitation prior to and after titration. No precipitation was ever observed. The ITC raw data was corrected for the heat of titrant dilution determined by an experiment conducted in an identical condition except that there was no protein in the sample cell. Since TA Instruments use a positive sign for an exothermic reaction, which is opposite to the conventional presentation used by MicroCal (GE), we manually converted the measured raw data that are presented in all figures.
Absorption measurements were carried out using a UV-1800 double-beam spectrometer (Shimadzu, Kyoto, Japan). The fluorescence spectra were recorded on a FluoroMax-3P (Horiba John Yvon, Inc.) equipped with excitation/emission polarizers and a temperature control unit. Since there is no Tyr residues in NCaBD, the Trp fluorescence spectra were obtained using 2-4 μM protein with an excitation wavelength of 280 nm at 20°C. The slit widths of 2 and 5 nm for excitation and emission, respectively, were chosen to eliminate photobleaching. For Ca2+ titration, solutions of Ca2+ ranging from 1 M to 1 μM were added in increments of 1 and 2 μL to 1 mL of the protein solution. After each addition of Ca2+, the sample was incubated for 1 min before measuring. All reported spectra were corrected for buffer effects, dilution factors, and any wavelength-dependent response for the fluorometer.
where F and Fo are the fluorescence intensities at a given concentration of quencher (Q) and in the absence of quencher, respectively; Ksv is the dynamic or collisional quenching constant.
Far-UV circular dichroism (CD)
The CD spectra were recorded using a JASCO J-715 instrument (JASCO Corporation, Japan) at ambient temperature. Approximately 0.1 mg/mL protein in 10 mM Tris, pH 7.5 in the presence of EDTA, Mg2+, or Ca2+ were placed in a cylindrical 0.1 cm path length quartz curve. All spectra were the average of 6 scans with a scan rate of 20 or 50 nm/min and were corrected by subtracting the spectra of buffers.
where ϴ is the fraction of the ligand-bound protein; Fmax and Fmin are the maximum and minimum intensities during titration; F is the intensity at a specific ligand concentration; [P]t and [L] are the concentration of total protein and free ligand (in a weak binding, the concentration of free ligand is approximately equal to the concentration of total added ligand).
ITC measures heat evolved (Q) from the formation of ligand-protein complex. This binding constant(s) and its corresponding molar heat enthalpy (ΔH) for the reaction are calculated directly from the plot of heat evolved for each injection (ΔQi) versus molar ratio of ligand and protein using the software associated with ITC. We used NanoAnalyze (TA Instruments) to fit our data with one- independent, two-independent, or cooperative binding models.
NCaBD containing two Ca2+ binding sites will need to be fit with a two binding site model. The linkage for a model-free binding and a model-dependent binding is shown in Figure 5, where the relationship of macroscopic (stepwise or stoichimetric) binding constants (K1 and K2), overall binding constants (β1 and β2) and microscopic association biding constants, KI and KII is shown.
Calcium binding domain
Flavin adenine dinucleotide
Isothermal titration calorimetry
Nicotinamide adenine dinucleotide phosphate
The N-terminal half of CaBD
We thank Dr. Carl Frieden at Washington University in St. Louis (WUSTL) for allowing us to use his CD spectrometer. We also thank the internal financial supports from Southern Illinois University Edwardsville: Seed Grants for Transitional and Exploratory Projects (STEP) to CW; Research Grants for Graduate Students (RGGS) to NR; and Undergraduate Research and Creative Activities (URCA) assistantship to CP. This work was supported in part by Cottrell College Science Awards 7322 from Research Corporation and National Science Foundation grant DUE-0941517 to CW.
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