Thermal Stability of NKX3.1 Constructs As Determined by CD Spectroscopy
The thermal stabilities of the NKX3.1 homeodomain-containing constructs with increasing amounts of the N-terminal sequence were determined by measuring the CD signal at 222 nm to monitor the changes in α-helix content as a function of temperature. The CD spectra of the NKX3.1 (1–184), (75–184), (97–184), and (114–184) constructs are consistent with the homeodomain containing significant α-helical content and the N-terminal region consisting of flexible, disordered structure (), as already shown in previous NMR studies (6
). Panels E and F of show the 222 nm CD signal and estimated unfolded population as a function of temperature. NKX3.1 (114–184) constructs with and without the His tag sequence (see Materials and Methods) yielded similar spectra and the same unfolding transition temperature within error (±1 °C), and shows the results for all constructs with the His tag retained. To mimic the phosphorylation of acidic domain residues Thr89 and Thr93, NKX3.1 (75–184) constructs with these residues either singly or doubly mutated to glutamate were also assessed. The CD spectra for the single and double mutants are nearly the same as that of the wild-type NKX3.1 (75–184) construct, and for the sake of clarity, only the curves for the double mutant are shown in . summarizes the unfolding transition midpoint temperatures for all the constructs, including the single mutants.
Figure 1 CD spectra and thermal unfolding curves. (A) NKX3.1 constructs (1–5) used for CD spectroscopy with a diagram showing the locations of the acidic domain (AD) and SRF-interacting (SI) motifs and homeodomain (HD). (B) Peptide mixtures (a–d) (more ...)
Unfolding Transition Temperatures of NKX3.1 Constructsa
NKX3.1 (114–184), or the HD construct, and NKX3.1 (97–184), or the SI/HD construct, show the lowest unfolding transition temperature, 40 ± 1 °C. Addition of the AD motif to the NKX3.1 (75–184) construct provides a modest increase to 42 ± 1 °C; that of the single mutants increases to 43 ± 1 °C, and the double mutant has the highest transition temperature, 44 ± 1 °C. These increases in stability are small, barely greater than error, but they do support the notion that the AD influences the stability of the HD, and that AD phosphorylation should be expected to further influence HD stability.
To confirm the expected increase in stability due to interaction with phosphorylated AD, CD was performed on NKX3.1 (114–184) in the presence of the synthetic AD peptide with both threonines phosphorylated, and it was compared to the corresponding nonphosphorylated AD peptide. NKX3.1 has other potential CK2 phosphorylation sites beyond those in the AD (9
), and in vitro phosphorylation of the NKX3.1 constructs was not pursued. The spectra and unfolding curves are shown in . The spectrum and unfolding curve for the phosphorylated AD peptide alone are also shown for reference. The CD results for the nonphosphorylated AD peptide alone were similar. The spectrum of the phosphorylated AD peptide alone, in particular how its signal at 222 nm becomes more negative as the temperature increases, is consistent with polyproline II secondary structure (16
); however, additional structural characterization of the peptide, measuring HN–Hα J
coupling and 1
H NOESY for instance, would be required to confirm that the ensemble average secondary structure lies in the polyproline II region of the Ramachandran map. Because of the high concentrations used, 100 μ
M NKX3.1 (114–184) and 1.6 mM peptide, a shorter, 0.2 mm cuvette was used, and the signals contain more noise due to scattering. The results are summarized in .
The phosphorylated AD peptide does provide a 2 °C increase in HD stability compared to the nonphosphorylated peptide, in agreement with the double mutant result given above. However, the actual unfolding transition temperatures, 34 ± 1 and 36 ± 1 °C, for the nonphosphorylated and phosphorylated peptides, respectively, are significantly lower than that for NKX3.1 (114–184) alone. Since previous NMR results suggested both the AD and SI regions might interact with the homeodomain (7
), CD was also measured in the presence of a synthetic peptide containing both the AD and SI sequences. The resulting unfolding transition temperature (42 ± 1 °C) agrees with the value for the NKX3.1 (75–184) construct, indicating that both the AD and SI motifs are required for the stabilizing interactions with the homeodomain.
Interaction of the AD and SI Motifs with the Homeodomain by NMR
shows the overlaid 15N HSQC spectra of the 15N-labeled NKX3.1 (114–184) construct alone and in the presence of the phosphorylated AD peptide. Large chemical shift perturbations of up to 0.32 ppm are seen, evidence of specific interactions with the homeodomain. summarizes these chemical shift changes. Three of the largest changes, those of Ser150 and Arg176 backbone amide signals and the Arg175 Hε side chain signal, were monitored as a function of peptide concentration (), and a binding constant (Kd) of 0.5 ± 0.2 mM was determined by a least-squares fit of the data to the predicted binding curves (see Materials and Methods). Including additional residues did not alter the results or quality of the fit. shows the locations on the homeodomain of the resonances most highly perturbed by addition of the phosphorylated AD peptide. A spectrum was also acquired with 1.6 mM phosphorylated AD (data not shown) and displayed a pattern of additional perturbations not evident at lower concentrations, so this spectrum was not used for analysis. The cause of the anomalous perturbations is unclear; perhaps some additional mode of interaction occurs at higher peptide concentrations, though additional study would be needed to confirm this.
Figure 2 NMR spectra titrated with the phosphorylated AD peptide. (A) Overlaid 2D 15N HSQC spectra of 100 μM NKX3.1 (114–184) without (red) and with 0.8 mM phosphorylated AD peptide (black). The resonances most perturbed by addition of phosphorylated (more ...)
Figure 3 NKX3.1 HD amide chemical shift perturbations due to AD interactions. (A) NKX3.1 (114–184) with 0.8 mM phosphorylated AD peptide, with the y-axis on the left. (B) NKX3.1 (114–184) with 1.6 mM nonphosphorylated AD peptide, with the y-axis (more ...)
The experiment was repeated in the presence of the nonphosphorylated AD peptide (). The observed perturbations, while weaker (< 0.1 ppm), have a pattern quite similar to that of the phosphorylated AD perturbations, suggesting nonphosphorylated AD interacts specifically with the homeodomain in a similar manner, albeit with a weaker binding constant. No saturation of the chemical shift changes was apparent up to the maximum peptide concentration used, 1.6 mM, and therefore, the binding constant for nonphosphorylated AD must be weaker than this value.
The 2D 15N HSQC NMR spectra of two constructs, NKX3.1 (75–184) and NKX3.1 (114–184), were compared to identify the intramolecular interactions of the AD and SI motifs with the homeodomain (). The observed chemical shift changes are small, with only a few stronger than noise (~0.02 ppm), implying either predominantly nonspecific interactions or weak specific interaction. The spectrum of the ADSI peptide with NKX3.1 (114–184) was also obtained (). Though not identical, some of the shift changes resemble the intramolecular changes (); they both show the largest change for the Arg141 side chain Hε signal, for example. The difference in the intramolecular and ADSI-induced shift perturbations compared to the AD-induced perturbations supports the CD result above showing different stabilization properties for the AD and ADSI peptides.
Modeling of the Interaction of the AD with the NKX3.1 HD
Model structures of possible complexes of the AD and phosphorylated AD peptides with the NKX3.1 HD were docked using the EMAP module of CHARMM (17
), and molecular dynamics and minimization were performed using MacroModel (Schrödinger Inc.). The purpose of the models was twofold: to explore whether specific interactions, such as hydrogen bonds, might explain some of the observed chemical shift changes and to test whether the linker between the HD and AD is sufficiently long to accommodate the putative specific interactions.
Ten random peptide conformations of both the AD and the phosphorylated AD were docked individually in two orientations to the HD using EMAP (see Materials and Methods). The largest amide chemical shift changes due to AD interactions were observed for Arg175 side chain and Ser150 backbone amides, and in most of the docked complexes, hydrogen-bonded conformations between the peptide and Arg175 side chain were maintained during the unrestrained dynamics and minimization. They were not maintained for the backbone amide of Ser150, though hydrogen bonds with the side chain OH group were not uncommon. Panels A and B of show representative hypothetical models of the AD–HD and phosphorylated AD–HD complexes, respectively. Perhaps the significant chemical shift perturbation of the Ser150 backbone amide could be due to interaction with its side chain; another possibility is that the peptide disrupts transient N-capping interactions between Glu153 and Ser150 at the beginning of HD helix II (18
Figure 4 Example model structures of (A) the AD peptide and (B) the phospho-AD peptide docked to the NKX3.1 HD. The HD ribbons are shaded from red at the N-terminus to blue at the C-terminus. These two examples highlight possible hydrogen-bonded contacts between (more ...)
shows the ensemble of phosphorylated AD complexes with AD–HD linkers included. Regardless of whether the peptide N-terminus was closer to Ser150 or Arg175, there was no difficulty in generating the linker for any of the docked complexes. Were the linker region too short, this might help to explain the difference in the observed intra- versus intermolecular AD–HD chemical shift perturbations; however, the modeling clearly indicates that the linker is sufficiently long to accommodate the putative specific interactions.
Another possibility is that the peptide concentrations used in the NMR experiments (up to 1.6 mM) are not representative of the intramolecular interaction. Using polymer theory for flexible, disordered structure, an effective concentration of the covalently linked AD peptide relative to the HD can be approximated. The effective concentration is related to the probability of a polymer having zero end-to-end separation, given here using the wormlike chain model (19
) in the long chain limit:
where n is the number of monomers in the chain (29 residues between the AD and HD), d is the monomer length (3.85 Å), and p is the persistence length, which is a measure reflecting how quickly the chain direction becomes randomized, and is taken here to be four monomer lengths. This effective intramolecular concentration is comparable to the peptide concentrations used for NMR. Neither linker length nor effective concentration can explain the observed differences between intra- and intermolecular AD–HD interactions, which further supports the CD and NMR results suggesting SI motif interactions directly influence AD–HD interactions.