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

¹ Department of Biomedicine, University of Bergen, 5009-Bergen, Norway
² Department of Biochemistry, Universidade de Coimbra, 3000 Coimbra, Portugal
³ Departamento de Química Física, Facultad de Ciencias, 18071 Granada, Spain

Reprint requests to: Aurora Martínez, Department of Biomedicine, Jonas Lies vei 91, 5009-Bergen, Norway; e-mail: aurora.martinez{at}ibmb.uib.no; fax: 47-55586360.

(RECEIVED December 28, 2003; FINAL REVISION February 4, 2004; ACCEPTED February 9, 2004)

	Abstract

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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

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.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
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 -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

TOP Abstract Introduction Results Discussion Materials and methods References

 
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 C atoms of Arg13 and the C 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).

View larger version (20K): [in this window] [in a new window]   Figure 1. Representative trajectory graphs from MD simulations. Distances between the C 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.

View larger version (64K): [in this window] [in a new window]   Figure 2. 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.

View larger version (29K): [in this window] [in a new window]   Figure 3. 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) Wiunphosphorylated-Wiphosphorylated. Positive values indicate more favorable interaction for those groups in phosphorylated wt-PAH.

View larger version (16K): [in this window] [in a new window]   Figure 4. 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.

View larger version (17K): [in this window] [in a new window]   Figure 5. 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. , molar ellipticity at 222 nm.

View larger version (72K): [in this window] [in a new window]   Figure 6. 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

TOP Abstract Introduction Results Discussion Materials and methods References

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 W_{q - 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, 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 [PDB] ; Kobe et al. 1999) are not observed in the structure of the phosphorylated enzyme (PDB 1PHZ [PDB] ), which resembles the iron-coordination in the substrate (and cofactor) bound ternary complexes of PAH (Andersen et al. 2002).

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
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 K_m 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 (V_max/K_m[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 [PDB] and 1PHZ [PDB] ; 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 W_i, which can be used to estimate the total charge–charge interaction energy in the proteins (W_{q - q}):

(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.

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