Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2004 May;13(5):1219–1226. doi: 10.1110/ps.03595904

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Frederico Faria Miranda 1,2, Matthías Thórólfsson 1, Knut Teigen 1, Jose M Sanchez-Ruiz 3, Aurora Martínez 1
PMCID: PMC2286772  PMID: 15096628

Abstract

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge–charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13–Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.

Keywords: phosphorylation, phenylalanine hydroxylase, molecular dynamics simulations, conformational stability, electrostatic interactions, proteolysis

Posttranslational modification of proteins by phosphorylation is a widely used mechanism to control many cellular functions (Graves and Krebs 1999). The effects of phosphorylation can vary from subtle modulations of substrate access to long-range allosteric effects (for review, see Johnson and Lewis 2001). The enzyme phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of l-phenylalanine (L-Phe) to l-tyrosine mainly in the liver, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA; Wretborn et al. 1980; Kaufman 1993). The enzyme requires a nonheme iron, a pterin cofactor, and molecular oxygen for activity (Flatmark and Stevens 1999; Fitzpatrick 2000). Defects in the human enzyme are associated with the genetic disease phenylketonuria (Scriver et al. 1995; Erlandsen and Stevens 1999), which is one of the most important disorders of amino acid metabolism. The crystal structures of the unphosphorylated and phosphorylated regulatory domain have been solved (Kobe et al. 1999). However, these structures do not significantly assist in the elucidation of the mechanism for activation by phosphorylation because the first 18 residues in the structure, including the phosphorylation site at Ser16 and located at the autoregulatory sequence (residues 1–30), were not identified in the electron density (Kobe et al. 1999). Our previous modeling and mutagenesis studies on human PAH have indicated that phosphorylation causes a conformational change at the N-terminal tail (residues 1–18) triggered by the electrostatic interaction of the incorporated phosphate and Arg13 (Miranda et al. 2002). The conformational switch from the unphosphorylated to the phosphorylated form resulted in an increased accessibility of the substrate to the active site. In addition, the phosphorylated enzyme and the mimicking mutants S16D-PAH and S16E-PAH showed an increased α-helical content and resistance to tryptic proteolysis (Miranda et al. 2002). NMR spectroscopy has also shown that the mobile N-terminal region of PAH shows an increased ordered secondary structure upon phosphorylation (Horne et al. 2002). FRET measurements and molecular dynamics (MD) simulations performed on a 10-residue peptide around the MAP kinase substrate Ser-31 in the homologous enzyme tyrosine hydroxylase also indicate that phosphorylation causes the peptide backbone to adopt a compact structure away from the active site (Stultz et al. 2002). This change also appears to be initiated by the favorable electrostatic interaction established by the phosphoserine and the side chain of a neighboring arginine. Similarly, phosphorylation at Ser 16 of phospholamban by PKA promotes a coil-to-helix transition that modulates the structural coupling between the transmembrane and cytosolic domains of the protein, most probably through an electrostatic linkage between the phosphate group and the kinase-recognizing residue Arg13 (Li et al. 2003). Thus, it appears that for many proteins the regulatory effects exerted by phosphorylation rely on a conformational switch put forward by the formation of salt bridges between the phosphate group and adjacent positively charged residues.

In this work we have performed MD simulations in the modeled structure of the full-length unphosphorylated and phosphorylated enzyme in order to investigate if the rearrangement of the N-terminal end brought about by the electrostatic interaction between the phosphate and Arg13 is built up long range to a global conformational change in the rest of the enzyme. The stabilizing and destabilizing interactions in the resulting structures were analyzed by our implementation of the Tanford-Kirkwood model (Ibarra-Molero et al. 1999a; Sanchez-Ruiz and Makhatadze 2001). This is admittedly a very simple model, but it has been shown to predict qualitatively the effect of charge-reversal and charge-deletion mutations on the stability of several proteins (Sanchez-Ruiz and Makhatadze 2001). In addition, the structural information obtained from the MD simulated conformers has been validated by differential scanning calorimetry (DSC), thermal-dependent circular dichroism (CD), and fluorescence spectroscopy using unphosphorylated and phosphorylated human PAH and the mutants S16D-PAH and S16E-PAH.

Results

MD simulations and charge–charge interactions calculations

The full-length models for both unphosphorylated and phosphorylated PAH at Ser16 (Miranda et al. 2002), which were previously prepared by using the anchor search grow method of DOCK 4.0 (Leach and Kuntz 1992), were subjected to MD simulations. The systems studied were well behaved, and no swelling was encountered during the simulations (Fig. 1 ▶). The root mean square (RMS) deviations of all heavy atoms with respect to the starting structure after equilibration were 3.0 Å and 3.5 Å for the unphosphorylated and phosphorylated forms, respectively, and were stable (± 0.3 Å) for the rest of the 600-psec simulations. Representative trajectory graphs showing the stability of the systems after 150 psec are shown in Figure 1 ▶, A and B. The displacement of the N-terminal tail on phosphorylation is illustrated by the 4-Å-increased distance between the Cα atoms of Arg13 and the Cα of Glu381 at the catalytic domain (Fig. 1A ▶). This displacement seems to be triggered by the electrostatic interactions established between Arg13 (and Lys14) and the phosphoserine (Fig. 2 ▶; Miranda et al. 2002), and is not accompanied by changes in the relative topologic rearrangement of the regulatory and catalytic domains, as exemplified by the similar distances between Trp120 (at the hinge between the regulatory N-terminal domain) and Trp326 (at the catalytic C-terminal domain) in the two enzyme forms (Fig. 1B ▶). This is in contrast to results obtained with activating mutations and incubation of PAH with L-Phe, for which the larger conformational changes affecting the relative orientation of the domains result in the exposure of Trp120 to the solvent (below; Thórólfsson et al. 2003).

Figure 1.

Figure 1.

Open in a new tab

Representative trajectory graphs from MD simulations. Distances between the Cα atoms of Arg13 (at the N-terminal autoregulatory sequence) and Glu381 (at the catalytic domain; A) and of Trp120 (at the hinge between the regulatory and catalytic domains) and Trp326 (at the catalytic domain; B) for unphosphorylated (dotted line) and phosphorylated (continuous line) PAH.

Figure 2.

Figure 2.

Open in a new tab

Overall structure of the full-length unphosphorylated and phosphorylated subunit of PAH. The 18-residue N-terminal tails of the unphosphorylated and phosphorylated forms are shown as purple and blue ribbons, respectively. (Insets) Detailed views of the average energy-minimized conformer over the last 50 psec of the 600-psec simulation for the unphosphorylated (purple backbone, top) and phosphorylated (blue backbone, bottom) PAH. The iron atom is shown in yellow.

Based on the final structural models obtained after MD simulations (Fig. 2 ▶), an analysis of the energy of the charge–charge interactions was performed in the dimeric forms. Most charged groups show negative 〈Wi〉 values, due to interactions mainly with groups of the opposite charge (Fig. 3 ▶). The resulting 〈Wq - q〉 values are negative for both enzyme forms, namely, −115 and −126 kJ/mole for the unphosphorylated and phosphorylated subunit PAH, respectively. 〈Wq - q〉 is considered to be an approximation of the charge–charge contributions to the unfolding Gibbs energies (−ΔGq - q), and our results indicate that the phosphorylated form is more stable. Nevertheless, a substantial contribution to the Δ〈Wq - q〉 =−16 kJ/mole per subunit between the phosphorylated and unphosphorylated form seems to arise mainly from the favorable interactions that Arg13 and Lys14 establish with the phosphate at Ser16 (Fig. 3B ▶), suggesting that the stabilization is not transmitted to other regions but is localized around the phosphate at the N-terminal tail. These local attractions seem to play a major role in the phosphorylation-induced–activating conformational change at the N-terminal tail, which exposes the active site (Fig. 2 ▶). In addition, the repulsion of the phosphate by Glu280, a residue that is further decompensated electrostatically in the phosphorylated form, and the favorable interaction of Arg13 with Asp145 also seem to play an important role in the formation of the phosphorylated structure (Figs. 2 ▶, 3B ▶). The electrostatic calculations also reveal the change from negative stabilizing to positive-destabilizing interactions that Glu353 and Glu381 experience upon phosphorylation (Figs. 2 ▶, 3B ▶). These two residues form salt bridges with Arg13 and Lys14 in the unphosphorylated form (Fig. 2 ▶; Miranda et al. 2002). Interestingly, the calculations further expose that phosphorylation results in a more favorable electrostatic compensation of residues Arg270 and His285, which are involved in direct interactions with the substrate (Teigen et al. 1999; Andersen et al. 2002). His 285 is in addition one of the coordinating residues to the iron.

Figure 3.

Figure 3.

Open in a new tab

Energy of charge–charge interactions for individual ionizable residues. (A) Unphosphorylated (filled circles) and phosphorylated (empty circles) PAH. Each value represents the energy of interaction of each group with all the other charges in the protein (〈Wi〉). A positive value for this interaction energy means that the group is predominantly involved in destabilizing interactions with charges of the same sign. Conversely, a negative value implies that the group is involved in predominantly stabilizing interactions with groups of the opposite charge. Calculations are made from the model structures in Fig. 2 ▶. (B) 〈Wi〉unphosphorylated-〈Wi〉phosphorylated. Positive values indicate more favorable interaction for those groups in phosphorylated wt-PAH.

L-Trp emission fluorescence

To probe the results from MD indicating a local nature of the conformational changes induced by phosphorylation, we studied the intrinsic fluorescence spectrum of unphosphory-lated wild-type PAH (wt-PAH), phosphorylated wt-PAH, S16D-PAH, and S16E-PAH. L-Trp to L-Phe scanning mutagenesis has shown that Trp120 is largely responsible for the fluorescence properties of the enzyme; namely, this residue accounts for ~61% of the total tryptophan fluorescence of PAH (Knappskog and Haavik 1995). In addition, the increase in fluorescence intensity and emission maximum that accompanies activation of the enzyme by L-Phe has been found to originate mainly from changes in the emission of Trp120 (Knappskog and Haavik 1995). This residue becomes more solvent-exposed in the substrate-activated enzyme, most probably as a result of domain movements around the hinge region Arg111–Thr117 (Thórólfsson et al. 2003). No significant changes were detected on the emission wavelength or fluorescence intensity upon phosphorylation of wt-PAH at Ser16 (Fig. 4 ▶). The phosphorylation-mimicking mutants S16D-PAH and S16E-PAH also exhibited similar fluorescence spectra as did phosphorylated wt-PAH (Fig. 4 ▶).

Figure 4.

Figure 4.

Open in a new tab

Intrinsic fluorescence emission spectroscopy. Emission spectra of unphosphorylated (dotted line) and phosphorylated (continuous line) wt-PAH. Protein samples were diluted to a final concentration of 1 μM in 20 mM Na-Hepes, 0.2 M NaCl (pH 7.0). The excitation wavelength was 295 nm, and the measured emission maxima were 337.5 nm (unphosphorylated PAH) and 337.1 nm (phosphorylated PAH). The mutants S16D-PAH and S16E-PAH showed similar emission spectra as phosphorylated wt-PAH.

CD and DSC

A further analysis on the plausible stabilizing effects of phosphorylation on the conformational stability of PAH was performed by thermal-dependent CD and by DSC. We have recently studied the thermostability of unphosphorylated human PAH by both CD and DSC (Thórólfsson et al. 2002). The thermal denaturation of the enzyme is characterized by two partially overlapping transitions with midpoint transition temperatures (Tm) of 46°C and 54°C, corresponding to the denaturation of the regulatory and catalytic domains of the enzyme, respectively. The phosphorylation at Ser16 did not result in a significant change of the Tm values for either transition as seen in the CD denaturation profiles (Fig. 5 ▶; Table 1). However, the incorporation of the phosphate group increases the negative ellipticity at 222 nm (Fig. 5 ▶), corresponding to a slightly higher α-helical content and a concomitant decrease of the unordered structure in the N-terminal tail (Fig. 2 ▶; Miranda et al. 2002). Corresponding DSC analyses on the two protein conformers (Table 1) and on the mutants S16D-PAH and S16E-PAH (data not shown) also confirmed that the increase in ordered secondary structure was not accompanied by a significant increase in thermostability. Moreover, phosphorylation or incorporation of the negatively charged residue at Ser16 did not have a significant effect on the calorimetric enthalpy or the van’t Hoff enthalpy values for the unfolding.

Figure 5.

Figure 5.

Open in a new tab

CD-monitored thermal denaturation. The samples were 10 μM subunit of unphosphorylated (dotted line) and phosphorylated (continuous line) wt-PAH prepared in 20 mM Na-phosphate and 0.15 M KF (pH 7.0). The scan rate was 0.7 K/min. θ, molar ellipticity at 222 nm.

Table 1.

Transition temperatures (Tm) for the thermal denaturation of unphosphorylated and phosphorylated wt-PAH monitored by thermal dependent CD and DSC

Unphosphorylated wt-PAH Phosphorylated wt-PAH
CD
    Tm1 (°C) 46.8 47.0
    Tm2 (°C) 54.4 54.6
DSC
    Tm1 (°C) 46.5 46.7
    Tm2 (°C) 55.2 54.6
Open in a new tab

The values are the average of two independent experiments.

Limited proteolysis

An indication that phosphorylation could increase the overall conformational stability of the PAH had been raised from our earlier results showing that phosphorylated wt-PAH, S16D-PAH, and S16E-PAH show increased protection against limited tryptic proteolysis (Miranda et al. 2002). However, the MD simulations and electrostatic calculations showing the local extent of the conformational changes effected by phosphorylation, as well as the CD and DSC results showing a similar thermostability for all forms (Table 1), prompted us to perform a closer investigation of their resistance to proteolysis. We investigated the limited chymotryptic proteolysis of the unphosphorylated and phosphorylated wt-PAH and of the S16E-PAH mutant. Opposite to the observed phosphorylation-induced and S16E mutation–induced protection toward limited tryptic proteolysis, these modifications did not render any protection toward proteolysis of the enzyme by chymotrypsin (Fig. 6 ▶; data not shown).

Figure 6.

Figure 6.

Open in a new tab

Limited tryptic and chymotryptic proteolysis of unphosphorylated and phosphorylated PAH. SDS-PAGE of the tryptic (A) and chymotryptic (B) products of unphosphorylated wt-PAH, and of the tryptic (C) and chymotryptic (D) products of phosphorylated wt-PAH. The enzyme samples were incubated for 20 min at 30°C at different trypsin and chymotrypsin protein ratios (μg : μg), namely, 0 : 1 (lane 2), 0.01 : 1 (lanes 3,5), and 0.1 : 1 (lanes 4,6). (Lane 1) Low-molecular-mass protein standards, namely, ovalbumin (45 kD) and carbonic anydrase (31 kD).

Discussion

About all known intracellular signaling pathways use protein phosphorylation to originate signals and to administrate them further (Graves and Krebs 1999). There seems to be no general molecular mechanism for control by phosphorylation, but instead a series of different responses with varying structural effects (Johnson and Lewis 2001). The modeled structure of full-length phosphorylated PAH indicates that this posttranslational modification induces a local conformational change brought about by the electrostatic interaction between Arg13/Lys14 and the phosphorylated Ser16, resulting in a more “open” active site, in agreement with a facilitated access of the substrate (Miranda et al. 2002). The analysis and optimization of the modeled structure by MD simulations performed in this work supports the validity of this hypothesis. Phosphorylation studies with other proteins have also shown that the incorporated phosphate often forms a network of hydrogen bonds with adjacent positively charged arginine residues, and in some cases, this local rearrangement of the residues is communicated to other distant regions in the protein (Johnson 1992; Lin et al. 1996; Russo et al. 1996; Stultz et al. 2002; Li et al. 2003). As found in this work for PAH, in many cases one of the adjacent interacting arginine residues belongs to the kinase recognition sequence, indicating the possibility that the structural changes induced by phosphorylation could often be initiated by common mechanisms. On the other hand, analysis of the MD-converged structure for the unphosphorylated and phosphorylated PAH forms show that no major overall conformational changes are observed upon incorporation of phosphate, as also indicated by the similar fluorescence emission spectrum for both forms of the protein. The emission of Trp120 is very sensitive to subtle changes in the solvent accessibility of the hinge region (Arg111-Thr117) between the catalytic and regulatory domain. Other activating mechanisms of the enzyme, for example, by incubation with N-ethylmaleimide, lysolecithin, or L-Phe, as well as mutagenesis at specific residues, result in a more solvent-exposed Trp120 and a red-shifted fluorescence spectrum (Phillips et al. 1984; Knappskog and Martínez 1997; Thórólfsson et al. 2003). Thus, our results are compatible with the absence of a solvent exposure of Trp120 or a generalized protein conformational change upon phosphorylation other than a shift of the backbone direction for the N-terminal tail of the enzyme with concomitant increase of the apparent α-helical structure and an increased affinity for the substrate (Miranda et al. 2002). On the other hand, the cooperative binding of the substrate certainly induces large conformational changes affecting the secondary, tertiary, and quaternary structure of the enzyme (Kaufman 1993; Thórólfsson et al. 2003).

Several proteins, such as the botulinum neurotoxins, have been shown to increase their stability upon phosphorylation (Encinar et al. 1998; Blanes-Mira et al. 2001). Phosphory-lated PAH, however, does not show increased thermal stability, despite the estimated Δ〈Wq - q〉 =−16 kJ/mole per subunit between the phosphorylated and unphosphorylated form. This discrepancy may reflect the limitations of the simple electrostatic model used, such as the facts that denatured-state interactions are not taken into account and only charge–charge interactions are considered (i.e., charge-solvation effects, which may be important, are not included). This notwithstanding, our simple electrostatic calculations do seem to be consistent with the electrostatic effect of phosphorylation being local (the significant change upon phosphorylation in the calculated charge–charge interaction energy arises mainly from the interaction between the phosphate and the nearby residues Arg13 and Lys14). Thus, the calculations do not suggest large conformational changes upon phosphorylation and, in this sense, seem consistent with the similar thermal stability of unphosphorylated and phosphorylated PAH, and their similar rate of proteolytic degradation by chymotrypsin. In this context, the increased resistance toward limited tryptic proteolysis exhibited by the phosphorylated form appears noteworthy. Our earlier results have shown that phosphorylation protects toward the specific cleavage at the carboxyl site of Arg13 most probably due to the inability of the protease to bind to this recognition site when it is in turn interacting with the phosphoserine (Døskeland et al. 1996; Miranda et al. 2002). This inability seems to decelerate the overall tryptic proteolysis rate in the rest of the protein. It is tentative to speculate on the physiological implications for a specific protection of phosphorylation toward degradation and turnover in vivo by proteases with trypsin-like specificity. The protection against cellular degradation shown by both phosphorylated ATF2 transcription factor (Fuchs et al. 2000) and the p27 (Kip1) protein (Ishida et al. 2000) has been associated to a protection from ubiquitination and decrease of degradation by the ubiquitin-dependent proteasome pathway. Although it has been established that human PAH is a target for ubiquitination by ubiquitin-conjugating enzyme system isolated from rat liver (Døskeland and Flatmark 2001), nothing is known about the effects of phosphorylation on ubiquitination of the enzyme and vice versa. Other studies have linked the higher intracellular stability of phosphorylated proteins, for example, α II spectin (Nicolas et al. 2002) to its protection toward the nonlysosomal neutral thiol protease calpain with partial trypsin-like cleavage specificity (Yajima and Kawashima 2002). It seems plausible that the local specific conformational change induced by phosphorylation of Ser16 at the autoregulatory sequence has consequences on the turnover of the enzyme in vivo, in addition to the short-term activation (~2.4 increased catalytic efficiency for the basal activity of the enzyme) and the synergy with L-Phe activation. With respect to this activation, the role of the local conformational change increasing the accessibility of the active site might be reinforced by the favorable effect that phosphorylation has on the energy of the charge–charge interactions of Arg270 and His285, residues directly involved on the binding of the substrate. A direct effect of phosphorylation on His285 can also be a way to positively modulate the reactivity of the coordinated nonheme iron and activate the enzyme, which would explain the higher catalytic efficiency of the phosphorylated enzyme even prior to activation by incubation with L-Phe (Miranda et al. 2002). In this context, it is interesting to note that two of the three iron-coordinated water molecules in unphosphorylated resting PAH (PDB 2PHM; Kobe et al. 1999) are not observed in the structure of the phosphorylated enzyme (PDB 1PHZ), which resembles the iron-coordination in the substrate (and cofactor) bound ternary complexes of PAH (Andersen et al. 2002).

Materials and methods

Expression and purification of wt-PAH, S16D-PAH, and S16E-PAH phosphorylation of wt-PAH

Expression of the fusion proteins with maltose binding protein by affinity chromatography on amylose resin (New England Biolabs), cleavage by Enterokinase (EKMax from Invitrogen), using 20 units protease/mg of fusion protein and purification of the isolated tetrameric forms of the enzymes (~90% of total active protein), was performed as described (Martínez et al. 1995). Wt-PAH incorporated ~1 mole of phosphate per mole PAH when phosphorylated at Ser16 by PKA as described (Miranda et al. 2002). The basal specific activity (in the absence of full activation by L-Phe) and the Km value for L-Phe were 1100 nmole Tyr/min/mg and 154 μM, respectively, for unphosphorylated PAH and 1500 nmole Tyr/ min/mg and 86 μM, respectively, for phosphorylated PAH. The catalytic efficiency (Vmax/Km[L-Phe]) thus increases ~2.4-fold upon phosphorylation.

Limited proteolysis by trypsin and chymotrypsin

For the limited proteolysis, either TPCK-treated trypsin from bovine pancreas (Sigma) or type II chymotrypsin (Sigma) was used at a protease/PAH ratio of 0 : 1, 0.002 : 1, 0.005 : 1, 0.01 : 1, and 0.1 : 1, in a final volume of 45 μL. The concentration of the various forms of PAH was 0.1 μg/μL. The reaction was performed for 20 min at 30°C and quenched with SDS denaturation buffer (3-min treatment at 95°C). The samples were then analyzed by SDS-PAGE (performed on 10% [w/v] acrylamide gels at 15 mA/ gel). The gels were stained by Coomassie brilliant blue, dried, scanned, and further analyzed using the software Deskscan II (Hewlett-Packard Co.) and Phoretix 1D Plus (Nonlinear Dynamics Ltd).

Circular dichroism

CD measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco Peltier 423S element, for temperature control. Purified samples of the enzyme were prepared on a degassed solution of 20 mM Na-phosphate and 0.15 M KF (pH 7.0) at a concentration of 10 μM. Equimolar amounts of ferrous ammonium sulfate per enzyme subunit were added in order to convert some iron-free apoenzyme present in the samples into holoenzyme (Chehin et al. 1998; Thórólfsson et al. 2002). Quartz cells of 1-mm path length were used. Thermal denaturation was monitored by following the changes in ellipticity at 222 nm, at a scan rate of 0.7 K/min in the temperature range 30°C to 70°C. Analysis of the data was performed by using the Standard Analysis program provided with the instrument.

Fluorescence measurements

Measurements were performed on a Perkin-Elmer LS-50B luminescence spectrometer with a constant temperature cell holder, using 0.5-cm path-length quartz cells. Enzyme samples (1 μM) were prepared in 20 mM Na-Hepes, 0.2 M NaCl (pH 7.0). The excitation wavelength was 295 nm, and the excitation and emission slits were three and five, respectively. All spectra were corrected for blank emission.

Differential scanning calorimetry

Measurements were performed on a MicroCal VP-DSC differential scanning calorimeter (MicroCal Inc.) with cell volumes of 0.5 mL at the indicated scan rates. A 100 mM Na-Hepes buffer, 0.1 M NaCl (pH 7.0) was used in all experiments. Calorimetric cells were kept under an excess pressure of 30 psi to prevent degassing during the scan. Purified tetrameric PAH enzymes (30 μM per subunit) with equimolar amounts of ferrous ammonium sulfate per subunit were used. Further details about the calorimetric experiment and the calorimetric data processing can be found elsewhere (Ibarra-Molero et al. 1999b; Thórólfsson et al. 2002).

MD simulations

MD simulations were performed in the dimeric form of the modeled full-length unphosphorylated and phosphorylated PAH at Ser16 (Miranda et al. 2002), previously prepared by molecular docking of the 18 N-terminal residues into the crystal structure of rat PAH (PDB codes 2PHM and 1PHZ; Kobe et al. 1999), which lacks structural information about this N-terminal region. The structures were solvated in a water box, using the TIP3P model for water molecules extended at least 6 Å in each direction from the solute (25,000 water molecules included), and Na+ and counter ions were added by using the LEAP module of the AMBER 7 program (parm94 force field included). The system was first energy-minimized and then heated for 10 psec to 300 K, and the Particle Mesh Ewald method was used to calculate long-range electrostatic interactions. Following the heating step, the system was maintained at 300 K, and the MD simulation was computed at 1.0-fsec intervals for 600 psec, with frames collected every 5.0 psec. The nonbonded interaction cutoff was set to 8.0 Å. From each of the frames of the unphosphorylated and phosphorylated structures, atomic coordinates were collected for comparison.

Charge–charge interactions calculations

Calculations of the energies of charge–charge interactions were carried by using our implementation of the Tanford-Kirkwood model with the solvent accessibility correction of Gurd, as we have previously described in detail (Ibarra-Molero et al. 1999a; Sanchez-Ruiz and Makhatadze 2001). Dielectric constants of 4.0 and 78.5 were used for the protein and the aqueous solvent, respectively. In this work we have focused on the energy due to the charge–charge interaction of group i with the rest of the ionizable groups in the protein 〈Wi〉, which can be used to estimate the total charge–charge interaction energy in the proteins (〈Wq - q〉):

graphic file with name M1.gif (1)

Acknowledgments

The technical help of Randi Svebak and valuable discussions with João Barroso and Raquel Carvalho are greatly appreciated. This work was supported by A Fundação para a Ciência e a Tecnologia (F.F.M.), The Research Council of Norway, and a FEBS Short-Term Fellowship (M.T.).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Abbreviations

  • BH4, (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin

  • CD, circular dichroism

  • DSC, differential scanning calorimetry

  • MD, molecular dynamics

  • PAH, phenylalanine hydroxylase

  • PKA, cyclic AMP–dependent protein kinase

  • wt-PAH, wild-type phenylalanine hydroxylase.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03595904.

References

  1. Andersen, O.A., Flatmark, T., and Hough, E. 2002. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-l-alanine, and its implications for the mechanism of catalysis and substrate activation. J. Mol. Biol. 320 1095–1108. [DOI] [PubMed] [Google Scholar]
  2. Blanes-Mira, C., Ibañez, C., Fernandez-Ballester, G., Planells-Cases, R., Perez-Paya, E., and Ferrer-Montiel, A. 2001. Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue. Biochemistry 40 2234–2242. [DOI] [PubMed] [Google Scholar]
  3. Chehin, R., Thórólfsson, M., Knappskog, P.M., Martínez, A., Flatmark, T., Arrondo, J.L., and Muga, A. 1998. Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy. FEBS Lett. 422 225–230. [DOI] [PubMed] [Google Scholar]
  4. Døskeland, A.P. and Flatmark, T. 2001. Conjugation of phenylalanine hydroxylase with polyubiquitin chains catalysed by rat liver enzymes. Biochim. Biophys. Acta 1547 379–386. [DOI] [PubMed] [Google Scholar]
  5. Døskeland, A.P., Martínez, A., Knappskog, P.M., and Flatmark, T. 1996. Phosphorylation of recombinant human phenylalanine hydroxylase: Effect on catalytic activity, substrate activation and protection against non- specific cleavage of the fusion protein by restriction protease. Biochem. J. 313 409–414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Encinar, J.A., Fernandez, A., Ferragut, J.A., Gonzalez-Ros, J.M., DasGupta, B.R., Montal, M., and Ferrer-Montiel, A. 1998. Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation. FEBS Lett. 429 78–82. [DOI] [PubMed] [Google Scholar]
  7. Erlandsen, H. and Stevens, R.C. 1999. The structural basis of phenylketonuria. Mol. Genet. Metab. 68 103–125. [DOI] [PubMed] [Google Scholar]
  8. Fitzpatrick, P.F. 2000. The aromatic amino acid hydroxylases. Adv. Enzymol. Relat. Areas Mol. Biol. 74 235–294. [DOI] [PubMed] [Google Scholar]
  9. Flatmark, T. and Stevens, R.C. 1999. Structural insight into the aromatic amino acid hydroxylases and their disease-related mutant forms. Chem. Rev. 99 2137–2160. [DOI] [PubMed] [Google Scholar]
  10. Fuchs, S.Y., Tappin, I., and Ronai, Z. 2000. Stability of the ATF2 transcription factor is regulated by phosphorylation and dephosphorylation. J. Biol. Chem. 275 12560–12564. [DOI] [PubMed] [Google Scholar]
  11. Graves, J.D. and Krebs, E.G. 1999. Protein phosphorylation and signal transduction. Pharmacol. Ther. 82 111–121. [DOI] [PubMed] [Google Scholar]
  12. Horne, J., Jennings, I.G., Teh, T., Gooley, P.R., and Kobe, B. 2002. Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase. Protein Sci. 11 2041–2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ibarra-Molero, B., Loladze, V.V., Makhatadze, G.I., and Sanchez-Ruiz, J.M. 1999a. Thermal versus guanidine-induced unfolding of ubiquitin: An analysis in terms of the contributions from charge–charge interactions to protein stability. Biochemistry 38 8138–8149. [DOI] [PubMed] [Google Scholar]
  14. Ibarra-Molero, B., Makhatadze, G.I., and Sanchez-Ruiz, J.M. 1999b. Cold denaturation of ubiquitin. Biochim. Biophys. Acta 1429 384–390. [DOI] [PubMed] [Google Scholar]
  15. Ishida, N., Kitagawa, M., Hatakeyama, S., and Nakayama, K. 2000. Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability. J. Biol. Chem. 275 25146–25154. [DOI] [PubMed] [Google Scholar]
  16. Johnson, L.N. 1992. Glycogen phosphorylase: Control by phosphorylation and allosteric effectors. FASEB J. 6 2274–2282. [DOI] [PubMed] [Google Scholar]
  17. Johnson, L.N. and Lewis, R.J. 2001. Structural basis for control by phosphorylation. Chem. Rev. 101 2209–2242. [DOI] [PubMed] [Google Scholar]
  18. Kaufman, S. 1993. The phenylalanine hydroxylating system. Adv. Enzymol. Relat. Areas Mol. Biol. 67 77–264. [DOI] [PubMed] [Google Scholar]
  19. Knappskog, P.M. and Haavik, J. 1995. Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli. Biochemistry 34 11790–11799. [DOI] [PubMed] [Google Scholar]
  20. Knappskog, P.M. and Martínez, A. 1997. Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase. FEBS Lett. 409 7–11. [DOI] [PubMed] [Google Scholar]
  21. Kobe, B., Jennings, I.G., House, C.M., Michell, B.J., Goodwill, K.E., Santarsiero, B.D., Stevens, R.C., Cotton, R.G., and Kemp, B.E. 1999. Structural basis of autoregulation of phenylalanine hydroxylase. Nat. Struct. Biol. 6 442–448. [DOI] [PubMed] [Google Scholar]
  22. Leach, A.R. and Kuntz, I.D. 1992. Conformational-analysis of flexible ligands in macromolecular receptor-sites. J. Comput. Chem. 13 730–748. [Google Scholar]
  23. Li, J., Bigelow, D.J., and Squier, T.C. 2003. Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban. Biochemistry 42 10674–10682. [DOI] [PubMed] [Google Scholar]
  24. Lin, K., Rath, V.L., Dai, S.C., Fletterick, R.J., and Hwang, P.K. 1996. A protein phosphorylation switch at the conserved allosteric site in GP. Science 273 1539–1542. [DOI] [PubMed] [Google Scholar]
  25. Martínez, A., Knappskog, P.M., Olafsdottir, S., Døskeland, A.P., Eiken, H.G., Svebak, R.M., Bozzini, M., Apold, J., and Flatmark, T. 1995. Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases: Isolation and characterization of the wild-type enzyme. Biochem. J. 306 589–597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miranda, F.F., Teigen, K., Thórólfsson, M., Svebak, R.M., Knappskog, P.M., Flatmark, T., and Martínez, A. 2002. Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase: Kinetic and structural effects. J. Biol. Chem. 277 40937–40943. [DOI] [PubMed] [Google Scholar]
  27. Nicolas, G., Fournier, C.M., Galand, C., Malbert-Colas, L., Bournier, O., Kroviarski, Y., Bourgeois, M., Camonis, J.H., Dhermy, D., Grandchamp, B., et al. 2002. Tyrosine phosphorylation regulates α II spectrin cleavage by calpain. Mol. Cell. Biol. 22 3527–3536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Phillips, R.S., Parniak, M.A., and Kaufman, S. 1984. Spectroscopic investigation of ligand interaction with hepatic phenylalanine hydroxylase: Evidence for a conformational change associated with activation. Biochemistry 23 3836–3842. [DOI] [PubMed] [Google Scholar]
  29. Russo, A.A., Jeffrey, P.D., and Pavletich, N.P. 1996. Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat. Struct. Biol. 3 696–700. [DOI] [PubMed] [Google Scholar]
  30. Sanchez-Ruiz, J.M. and Makhatadze, G.I. 2001. To charge or not to charge? Trends Biotechnol. 19 132–135. [DOI] [PubMed] [Google Scholar]
  31. Scriver, C.R., Eisensmith, R.C., Woo, S.L.C., and Kaufman, S. 1995. The hyperphenylalaninemias in man and mouse. Annu. Rev. Genet. 28 141–165. [DOI] [PubMed] [Google Scholar]
  32. Stultz, C.M., Levin, A.D., and Edelman, E.R. 2002. Phosphorylation-induced conformational changes in a mitogen-activated protein kinase substrate: Implications for tyrosine hydroxylase activation. J. Biol. Chem. 277 47653–47661. [DOI] [PubMed] [Google Scholar]
  33. Teigen, K., Frøystein, N.Å., and Martínez, A. 1999. The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: Implications for the catalytic mechanism. J. Mol. Biol. 294 807–823. [DOI] [PubMed] [Google Scholar]
  34. Thórólfsson, M., Ibarra-Molero, B., Fojan, P., Petersen, S.B., Sanchez-Ruiz, J.M., and Martínez, A. 2002. l-Phenylalanine binding and domain organization in human phenylalanine hydroxylase: A differential scanning calorimetry study. Biochemistry 41 7573–7585. [DOI] [PubMed] [Google Scholar]
  35. Thórólfsson, M., Teigen, K., and Martínez, A. 2003. Activation of phenylalanine hydroxylase: Effect of substitutions at Arg68 and Cys237. Biochemistry 42 3419–3428. [DOI] [PubMed] [Google Scholar]
  36. Wretborn, M., Humble, E., Ragnarsson, U., and Engstrom, L. 1980. Amino acid sequence at the phosphorylated site of rat liver phenylalanine hydroxylase and phosphorylation of a corresponding synthetic peptide. Biochem. Biophys. Res. Commun. 93 403–408. [DOI] [PubMed] [Google Scholar]
  37. Yajima, Y. and Kawashima, S. 2002. Calpain function in the differentiation of mesenchymal stem cells. Biol. Chem. 383 757–764. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES