Conformational States and Kinetics of the Calcium Binding Domain of NADPH Oxidase 5

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA), and used without further purification. All buffers used in the protein purification and the subsequent spectroscopic characterization were rendered “Ca²⁺-free” by treatment with Chelex-100 (Bio-Rad Laboratories, Hercules, CA). The spectroscopic measurements were performed at least three times using at least three different protein batches and were reproducible.

Plasmid Constructions

The pLW vector, a generous gift from Dr. Lucy Waskell at the University of Michigan, was digested with Nco I and Xho I, and a phosphorylated double stranded oligonucleotide (5’-CATGGCGCATCATCACCATCACCATGCGGTAC and 5’- CGCATG GTGATGGTGATGATGCGC) that encodes peptide sequence MAHHHHHAV and contains Nco I and Kpn I compatible sites (underlined), flanking 5’ and 3’ ends, was ligated to the digested pLW vector to create a linear pLW-His₆ vector containing Kpn I and Xho I cloning sites. The cDNA encoding human NOX5β was a generous gift from Dr. Botond Bánfi at the University of Iowa. The CaBD domain (residues 1-184) was amplified by polymerase chain reaction (PCR) with the primers: 5’-GACTGGTACCG ATGAGTGCCGAGGAG) and 5’-CGCACTCGAGTTAGG CGCGGGTCAGCTG. The forward and reverse primers introduced two restriction sites (underlined) for Kpn I and Xho I sites on both ends and a stop codon in the 3’ end. The PCR product was subcloned into the above pLW-His₆ vector to create the plasmid pLW-His₆CaBD. The recombinant protein was constructed in such way that it contains an additional eleven amino acids, MAHHHHHHAVP, on its N terminus. The plasmid containing the mutant E99Q/E143Q was generated using a QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Two sequential site-directed mutations were performed by first generating the single mutation E99Q, and then E99Q/E143Q. The clones were sequenced to confirm the mutations and the oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA).

Recombinant Protein Expression and Purification

The plasmids pLW-His₆CaBD or pLW-His₆E99Q/E143Q were introduced into BL21(DE3) E. coli cells. Cells were grown in Luria−Bertani (LB) medium to OD₆₀₀ 0.5-0.7, and the expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 ^oC for 4 h. All of the following purification steps were performed at 4 ^oC. The histidine-tagged recombinant proteins were first purified with a Ni²⁺-chelating Probond column (Invitrogen Carlsbad, CA). Briefly, after harvest cells were resuspended in a buffer containing 50 mM Tris, pH 7.5, 0.5 M NaCl, 10 mM Imidazole (Buffer A) followed by homogenization and centrifugation. The resulting supernatant was loaded on a Probond column equilibrated with Buffer A. The column was washed with the same buffer until OD₂₈₀ reached baseline. The target protein was eluted with Buffer A containing 300 mM imidazole. Protein was further purified with a phenyl-sepharose column (GE Healthcare BioSciences, Piscataway, NJ) as previously reported for CaM [15]. The purified proteins were then buffer-exchanged in an Amicon Ultra centrifugal filter device (5,000 Da MWCO, Millipore, Bellerica, MA) with a buffer containing 50 mM Tris, 0.125 M NaCl, 5 % glycerol, 1 μM EDTA, pH 7.5 and stored at -80 ^oC until use. The protein purity was estimated > 95 % based on density profiles measured using UN-SCAN-IT software (Silk Scientific, Inc, Orem, UT).

Preparation of AEDANS-Labeled CaBD and E99Q/E143Q

Approximately 100 μM purified CaBD or E99Q/E143Q in 50 mM EPPS, pH 7.5 was incubated with 2 mM 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS, Invitrogen) in the presence of 1 mM Ca²⁺ at 4 ^oC overnight in the dark. Iodoacetamide was added to the reaction mixture (final concentration was 1 mM) to quench the reaction. The free dye and quencher molecules were removed by passing the mixture through a Ni²⁺-chelating column, and the AEDANS-labeled protein was recovered by eluting with a 0.3 M imidazole solution. The stoichiometry of the labeling was determined to be 0.93 ± 0.11 based on the protein assay kit (Bio-Rad Laboratories) for proteins and the absorption coefficient (5,700 cm^-1M^-1) for IAEDANS.

Spectroscopic Measurements

Absorption measurements were carried out using a Cary-3 UV/vis double-beam spectrometer (Varian, Inc., Lake Forest, CA). The fluorescence spectra were recorded on a FluoroMax-3P (Horiba John Yvon, Inc., Edison, NJ) equipped with excitation/emission polarizers and a temperature control unit. In all cases, the fluorescence spectra were typically obtained using 2-10 μM protein with the excitation wavelengths at 295, 340, and 350 nm for Trp, AEDANS, and ANS fluorescence, respectively, at 20 ^oC. The window widths of 2 and 5 nm for the entrance and exit slits were chosen to eliminate photobleaching. All reported spectra were corrected for buffer effects, dilution factors, and any wavelength-dependent response for the fluorometer.

Stern-Volmer Quenching

Intrinsic and extrinsic fluorescence quenching experiments were carried out at 20 ^oC by adding a stock solution of 6 M acrylamide, which was determined using the absorbance coefficient ε₂₉₅=2.5 M^-1 cm^-1, to the sample solution containing 4-10 μM protein and 50 mM Tris, 0.125 M NaCl, pH 7.5. The following Stern-Volmer quenching equation was used for fitting

where F and F_o are the fluorescence intensities at a given concentration of quencher and in the absence of quencher, respectively, K_sv is the dynamic or collisional quenching constant, and [Q] is the concentration of added quencher.

Far-UV Circular Dichroism (CD)

The CD spectra were recorded using a JASCO J-715 instrument (JASCO Corporation, Japan) equipped with a temperature control unit. CD spectra were recorded using a cylindrical 0.1 cm path quartz curve and were shown as the average of 3 scans using a spectra bandwidth of 1.0 nm. In all measurements, 0.1 mg/ml proteins in 10 mM Tris, pH 7.5 with 1 mM Ca²⁺, 10 mM Mg²⁺, or 0.5 mM EDTA were scanned from 190 nm to 260 nm.

Kinetics of Metal Dissociation

The kinetics was measured using the FluroMax-3P with a stopped-flow accessory SFA-20 (Tgk Scientific, Bradford-on-Avon, UK). To determine the calcium dissociation rate constant, a solution containing 5 to 10 μM unlabeled protein (or 2 to 5 μM labeled protein) and 100 μM Ca²⁺ (or 2 mM Mg²⁺ plus 1 mM EGTA) was rapid-mixed with a solution containing EDTA ranging from 10 to 20 mM EDTA at 20 ^oC. Ca²⁺ or Mg²⁺ dissociation was monitored by fluorescence decay. Six to eight injections were averaged to generate the final traces shown in Fig. (5). The integration time and time interval were 2 and 5 ms, respectively. Since the data acquisition for our instrument is triggered after the stopped syringe meets the stop block, the points collected in the original data set are tens of ms later after mixing. The following single-exponential decay equation was used to fit the data

where Y is the observed fluorescence intensity at time t, Y_o and A_o are variables, and k_off is the dissociation rate constant. The “dead time” x_o was determined independently [16], and can be estimated from fitting. In our setting, it was determined to be 20 ms.

Chemical Stabilities

To determine chemical stabilities, approximately 10 μM unlabeled or 5 μM AEDANS-labeled protein was incubated in a buffer containing 50 mM EPPS, pH 8.0, 500 μM EDTA (1 mM Ca²⁺ or 10 mM Mg²⁺), and guanidine hydrochloride (Gdn) ranging from 0 to 6 M for at least 6 h. The samples were excited at 295 nm (340 nm for the labeled proteins), and the wavelengths of their emission were recorded. The maximum emission wavelengths of AEDANS-CaBD and AEDANS-E99Q/E143Q induced by Gdn were fitted into eq.3 to determine the fraction of unfolded protein (f_U), assuming a two-state unfolding model

where F_N, F_D, and F_obs represent the maximum emission wavelengths in the native state, unfold state, and in the presence of a given concentration of the denaturant ([D]), respectively. Because F is linearly dependent on the Gdn concentration in the native and unfolded states, F_N and F_U were determined from the baselines prior and after the unfolding transition using where m_N and m_D are the slopes, and Y_N and Y_D are intercepts.

For chemical stability determined by Trp fluorescence, the f_u was simply calculated as

where the λ_obs is the maximum wavelength of the protein at specific concentration of Gdn, λ_0M and λ_6M are the maximum wavelengths of the protein in the absence of Gdn and in the presence of 6 M Gdn, representing the completely folded and unfolded states.

Effects of Metal Ions on the Fluorescence Spectra of CaBD

Previously Bánfi et al. had expressed the recombinant CaBD (residues 1-169) of NOX5 in E. coli, in which it was initially expressed as a glutathione S-transferase (GST) fusion protein followed by thrombin cleavage [10]. Since CaBD contains three tryptophan (W9, W12, and W165) and one tyrosine (Y87), it is suitable for a study using fluorometry. It had been demonstrated that its Trp fluorescence intensity was enhanced upon Ca²⁺ binding. Interestingly, the fluorescence of its half domains, termed as N-CaBD (residues 1-78) and C-CaBD (residues 79-169), did not respond Ca²⁺, and therefore it was concluded that the resulting fluorescence enhancement was only associated with the relative conformational change of the two half domains. To eliminate the cleavage step used in the previous study that limits the amount of purified protein for structural and functional studies, we placed a six-histidine tag at the N-terminus of CaBD and purified it to be homogeneous. Our approach generated a good amount of purified recombinant protein with typical yields of 10 – 20 mg per liter culture.

We then characterized our recombinant CaBD by performing similar experiments using fluorescence spectroscopy except that an excitation wavelength at 295 nm was used to selectively excite Trp residues. Ca²⁺ binding to CaBD enhanced the fluorescence ~40 %, as determined by the integration of the spectra (all subsequent cited enhancements were from integration) and the maximum peak was shifted from 340 nm to 344 nm (Fig. 1A). The calcium-induced fluorescence change indicates that our protein was functionally active, but the enhancement was somewhat lower than the previous report [10]. The reason for this discrepancy is not clear, but it may be due to the difference in the protein constructs such as the different additional N-terminal residues and a longer polypeptide we used (residues 1-184), excitation wavelengths, and/or purification procedures. We also detected significant fluorescence change upon Mg²⁺ binding though the enhancement was lower (~ 10 %). The signal leveled off after approximately 600 μM of Mg²⁺, but it is not clear whether all EF-hands were completely occupied at this concentration, mainly due to lack of information regarding the relationship between structural and spectroscopic changes. Therefore, 2 mM Mg²⁺ was used in our all subsequent experiments except chemical unfolding experiments to ensure saturation. The EF-hands of CaBD appeared to bind specifically to Ca²⁺ because the addition of 40 μM Ca²⁺ to Mg²⁺-bound CaBD quickly converted it to its Ca²⁺-bound form judged by the different fluorescence profiles of the metal-free and metal-bound forms. This finding is consistent with the previous result showing that Mg²⁺ does not affect Ca²⁺ binding to CaBD [10].

Since the intrinsic fluorescence measurements only reflect the changes near the vicinities of the three Trp residues, it is difficult to differentiate the fluorescence contribution from each individual residue. In order to obtain more specific structural information, we labeled the Cys107 residue with IAEDANS to generate AEDANS-CaBD. The Cys107 residue is located between the 3^rd EF-loop (residues 88-99) and 4^th EF-loop (residues 132-143) in the C-terminal half, and the labeling on this site should serve as a particular probe for the behavior of the C-terminal lobe. Ca²⁺ and Mg²⁺ binding to AEDANS-CaBD enhanced the fluorescence intensity by 18 % and 10 %, respectively, and their maximum emission wavelengths were shifted from 470 nm to 466 and 460 nm for Mg²⁺ and Ca²⁺ binding, respectively, which suggest the local environment of AEDANS is more hydrophobic in the Ca²⁺-bound form than in the Mg²⁺-bound form (Fig. 1B). In our case, fluorescence spectra of Mg²⁺-CaBD were identical in the presence and absence of EGTA, a specific Ca²⁺ chelating agent, indicating such signal enhancement upon Mg²⁺ addition was not due to calcium contamination. Despite the fact that such a change can be monitored by fluorescence spectroscopy, the lack of a direct stoichiometric relationship for the amount of Ca²⁺ or Mg²⁺ binding that truly affects Trp and AEDANS fluorescence prevents us determining Ca²⁺/Mg²⁺ binding affinity. The increases in Trp and AEDNAS fluorescence caused by Mg²⁺ binding were smaller than those caused by Ca²⁺ binding, which indicates the local environments of Trp and Cys107 residues were different among the apo, Ca²⁺- and Mg²⁺-bound forms.

ANS Binding and Stern-Volmer Quenching

The dye ANS was used to investigate the solvent-accessible hydrophobic surface in the apo-, Ca²⁺- and Mg²⁺-bound states (Fig. 2). Similar to the previous result using TNS [10], Ca²⁺ binding to CaBD caused ~ 4 fold increase in ANS fluorescence, but Mg²⁺ binding to CaBD slightly increased ANS fluorescence. These data indicate that Mg²⁺-CaBD has a structure similar to the apo form. To further test this hypothesis, we determined the accessibility of the Trp residues and the AEDANS motif by fluorescence quenching. Fig. (3) shows the Stern-Volmer plots for the acrylamide quenching. Intrinsic fluorescence quenching revealed an upward quenching curve, which was more obvious with high concentration of quencher (data not shown). The upward curves are consistent with ground state heterogeneity and occurrence of dynamic and static quenching, but it is difficult to analyze those effects due to lack of structural information. In order to easily compare Trp accessibility, we only determined the quenching in the low concentrations of acrylamide and fitted the data with a linear quenching equation to obtain the Stern-Volmer quench constant (K_sv). The structures of the individual forms can be differentiated by fluorescence quenching: Trp residue(s) were more accessible for quenching in the calcium-bound form with the K_sv value of 9.49 M^-1, than the Mg²⁺-bound form with the K_sv value of 5.04 M^-1 and the apo form with the K_sv value of 5.28 M^-1 (Table 1). AEDANS fluorescence quenching shows a similar trend, but the Mg²⁺-bound form appeared to be more sensitive than Trp quenching. This suggests that Mg²⁺ binding causes structural changes in the C-terminal half, but the change does not induce exposure of hydrophobic patches for ANS binding or enhance Trp fluorescence quenching. Both intrinsic and extrinsic fluorescence quenching revealed that there is an increase in the exposure of chromophores upon Ca²⁺ binding, whereas Mg²⁺ only affects the C-terminal half. It is not clear whether the N-terminal half binds Mg²⁺ because there is no suitable fluorophore for detection of Mg²⁺ binding at that half domain.

Secondary Structures Determined by Circular Dichroism

The studies using fluorescence spectroscopy suggest significant conformational changes in CaBD upon Ca²⁺ binding. To further determine such change, we used CD spectroscopy to monitor the secondary structure content. In the absence of Ca²⁺, the CD spectrum shows two negative elliptical peaks near 208 and 222 nm and one large positive band at 190 nm (Fig. 4), which are characteristic peaks of proteins containing a dominate α-helical secondary structure. However, neither the addition of Ca²⁺ nor Mg²⁺ significantly changed the CD signal. These data are similar to the studies showing that the secondary structures of the CaM superfamily are nearly unaltered by Ca²⁺ or Mg²⁺ binding, but that the angle and distances between structural motifs change [17].

Kinetics of Calcium and Magnesium Association and Dissociation

We used fluorescence spectroscopy coupled with a stopped-flow device to measure the kinetics of the calcium and magnesium association and dissociation of CaBD. To measure calcium association, a solution containing CaBD in the presence of 10 μM EDTA was rapid-mixed with an equal volume of a buffer containing Ca²⁺ ranging from 50 to 200 μM. The calcium binding was then monitored by fluorescence. In all cases, the calcium binding was too fast to be detected in our stopped-flow spectrometry (data not shown). This fast binding was confirmed by an experiment including ANS in the reactant syringes in the same condition, in which ANS fluorescence increased and reached the maximum at 75 ms (data not shown), thus indicating a slow ANS binding after the fast calcium binding. To trigger the calcium dissociation from CaBD, a large excess amount of EDTA was mixed with Ca²⁺-CaBD. The kinetic traces obtained fit well to a single-exponential equation (Fig. 5B) and the dissociation rate constant (k_off) was determined to be 4.5 ± 0.3 s^-1 (mean ± SD, n = 5). The measured kinetic trace was identical to the experiment using AEDANS-CaBD performed in the same condition, indicating that dye-modification did not impair calcium binding, or at least calcium dissociation. However, the amplitude of the fluorescence decay was approximately 55 - 65% of total Trp or AEDANS intensity changes (i.e. difference between the Ca²⁺- and apo-CaBD), in which the initial intensity for the slow decay was estimated by extrapolating the fitted curve to the time before mixing (i.e. -20 ms), thus implying that there was a non-detectable fast decay. Since AEDANS was labeled specifically in the C-terminal half, it is reasonable to conclude that the slow phase represents the calcium dissociation from the C-terminal half, whereas the fast phase is associated with calcium dissociation from the N-terminal half, in which the cys107-labeled AEDANS is able to sense such a change. To test this hypothesis, we generated a double mutant, E99Q/E143Q, in which the calcium binding sites in the 3^rd and 4^th EF-hand motifs were removed. We changed the most frequently occurring negative residue, glutamate, at position 12(-Z) to glutamine in its canonical EF-hand motifs because several calcium binding proteins demonstrate that such mutation impairs the calcium binding. Such mutational changes were expected to have minimal overall alternation on protein structure [18]. The mutant’s partial inability to bind Ca²⁺ was proved by fluorescence quenching in which its K_sv values were almost identical in all states (Table 1), and by the chemical stability discussed below (Fig. 6). Using the unlabeled and labeled E99Q/E143Q, the slow fluorescence decay previously observed in the wild type CaBD disappeared, displaying only a non-detectable fast fluorescence decay (Fig. 5A). Using the dissociation constant for the C-terminal half, K_d,C = 4 μM and the equation K_d,C = k_off,C/k_on,C, the association rate constant (k_on,C) is calculated to be 1.1 x 10⁶ M^-1 s^-1. If we assume that the fast decay is within the dead time (i.e. 20 ms), the dissociation rate constant for the N-terminal half, k_off,N, is estimated to be > 300 s^-1, therefore giving k_on,N > 1.5 x 10⁷ M^-1 s^-1 using K_d,N = 20 μM. The lower K_d value for Ca²⁺ binding to the N-terminal half is mainly from its fast calcium dissociation. The data obtained from the previous study examining binding affinity [10] and our kinetics on CaBD is very similar to CaM, in which its C-terminal half was shown to have a tighter binding affinity and a slower calcium dissociation [19].

While the decay of Trp fluorescence can be easily explained due to the relative changes of the two half domains, the decrease in AEDANS fluorescence, implied in the first calcium dissociation (~ 40 % of amplitude), does suggest a significant interaction between the dye and the N-terminal half, possibly through spatial contact and/or the interdomain link. This argument was supported by the fact that the fluorescence spectrum of E99Q/E143Q showed that this mutant still responded to Ca²⁺ binding with 28 and 9 % enhancements (40 and 18 % for the wild type) for Trp and AEDANS fluorescence, respectively (Fig. 1C & D). The intrinsic fluorescence change for this mutant might be due to the relative change of two rigid halves, i.e. Ca²⁺-N-CaND and apo-C-CaBD, by which the interface formed through such interaction maintains a weaker ANS binding (Fig. 2). Such an explanation could not be simply applied to AEDANS-E99Q/E143Q. It has been seen that similar mutations on the 1^st or 2^nd EF-hands in the N-half domain of CaM alter structure, and thus fluorescence, resulting in Ca²⁺-binding to the C-terminal half domain of CaM, but not Ca²⁺ binding itself [20]. Furthermore, the possibility of the C-terminal half of this mutant still retaining calcium binding capacity in our experimental condition was eliminated because same mutation on C-CaBD (residues 79-184) resulted in the losing of AEDANS fluorescence response upon Ca²⁺ binding (unpublished results). Therefore, the calcium binding strengths of the N-terminal half and the C-terminal half are independent as reported previously [10], but their spectroscopic behaviors and thus their structures, appear not to be completely independent. Due to cross communication between the two half domains, our results also raise the question of whether calcium binding within and between the half domains is non-cooperative as previously reported [10].

The kinetics of Mg²⁺ association was very similar to that of Ca²⁺. The Mg²⁺ association was too fast to be detected. The dissociation of Mg²⁺ triggered by EDTA addition was only observed using AEDANS-CaBD (Fig. 5C). The dissociation rate constant was determined to be 6.65 ± 1.02 s^-1. The amplitude of such decay was identical to the intensity change determined from the steady states, which suggests that this Mg²⁺ dissociation is from the C-terminal lobe. This conclusion is supported by the fact that AEDANS-E99Q/E143Q did not respond to Mg²⁺ even at very high concentration of Mg²⁺. Interestingly, the Mg²⁺ dissociation from CaBD was too fast to be detected by Trp fluorescence.

Chemical Stabilities of CaBD and Mutant

Since the data from the previous result indicates structural independence of the N- and C-terminal halves, the stability of the individual domains may be different in relation to their thermodynamics and kinetics. We therefore performed chemical unfolding induced by guanidine hydrochloride (Gdn). Fig. (6A) shows a plot of the fractions of unfolded protein as a function of Gdn concentrations. The unfolding of CaBD monitored by intrinsic fluorescence was not a two-state unfolding model. This is consistent with the fact that the three Trp residues are near the peptide ends where structures tend not to be compact. In the presence of Ca²⁺, the overall stability was enhanced. There were two obvious transitions at ~ 1 and 4.5 M Gdn with an intermediate at 2-3 M Gdn. The calcium-bound CaBD is substantially more stable than the apo form, and the result agrees with the prediction from the theory of ligand binding [21]. Unfortunately, there is no constructive information that can be gathered from intrinsic fluorescence. To acquire specific structural stability, the same experiment using the cys107-labeled CaBD was conducted. Increasing Gdn concentration resulted in red shift in wavelengths from 470 to 496 nm in the absence of Ca²⁺ (data not shown); the unfolding curve, corrected for denaturant effects, appeared to be a two-state model. Fig. (6B and 6C) show a plot of the fraction of unfolded protein as a function of denaturant, and the parameter C_m was determined at the concentration of Gdn where 50% of the protein was induced unfolded. The unfolding monitored by the extrinsic fluorescence gave the C_m values approximately 2.5 M and 4.7 M in the absence and presence of Ca²⁺, respectively; the latter being consistent with the second transition obtained from intrinsic fluorescence. The data also indicated that the C_m values of the apo forms of N-terminal and C-terminal halves were near 2.5 M. Ca²⁺ binding most likely increases the stability of the C-terminal half, but not the N-terminal half. This conclusion was supported by using AEDANS-E99Q/E143Q. The unfolding curves were identical in the presence of EDTA, Mg²⁺, and Ca², consistent with our previous fluorescence quenching result showing inability of calcium binding in the C-terminal half. The unfolding study of Ca²⁺-bound AEDANS-E99Q/E143Q also suggests that the C_m value for Ca²⁺-bound N-terminal half is ~2.5 M. Interestingly, Mg²⁺ appeared to stabilize CaBD, more specifically the C-terminal half. This unfolding is concentration-dependent (data not shown) because lowering Ca²⁺ or Mg²⁺ concentration destabilizes CaBD as seen in CaM [21]. It appears that the domain that has a higher calcium binding affinity has a higher degree of stability. This is seen for the C-terminal half, in which its higher chemical stability is due to a higher unfolding free energy difference (ΔG_NU) between the apo and calcium-bound states. In contrast, the N-terminal half domain has free energies which are comparable in apo and calcium-bound forms, thus lowering ΔG_NU. More thorough unfolding studies with the individual halves using appropriate labeling or other techniques will further determine their free energy difference and cooperative unfolding transition for each domain. Our result indicates that the correlation between binding affinity and domain stability for CaBD agrees with other calcium binding proteins [20, 22].