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• W2895679935 abstract "Phenylalanine hydroxylase (PAH) regulates phenylalanine (Phe) levels in mammals to prevent neurotoxicity resulting from high Phe concentrations as observed in genetic disorders leading to hyperphenylalaninemia and phenylketonuria. PAH senses elevated Phe concentrations by transient allosteric Phe binding to a protein–protein interface between ACT domains of different subunits in a PAH tetramer. This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. To investigate this allosteric sensing mechanism, here we used the GROMACS molecular dynamics simulation suite on the [email protected] computing platform to perform extensive molecular simulations and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. These simulations strongly implicated a conformational selection mechanism for Phe association with ACT domain dimers and revealed protein motions that act as a gating mechanism for Phe binding. The MSMs also illuminate a highly mobile hairpin loop, consistent with experimental findings also presented here that the PAH variant L72W does not shift the PAH structural equilibrium toward the activated state. Finally, simulations of ACT domain monomers are presented, in which spontaneous transitions between resting-state and activated conformations are observed, also consistent with a mechanism of conformational selection. These mechanistic details provide detailed insight into the regulation of PAH activation and provide testable hypotheses for the development of new allosteric effectors to correct structural and functional defects in PAH. Phenylalanine hydroxylase (PAH) regulates phenylalanine (Phe) levels in mammals to prevent neurotoxicity resulting from high Phe concentrations as observed in genetic disorders leading to hyperphenylalaninemia and phenylketonuria. PAH senses elevated Phe concentrations by transient allosteric Phe binding to a protein–protein interface between ACT domains of different subunits in a PAH tetramer. This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. To investigate this allosteric sensing mechanism, here we used the GROMACS molecular dynamics simulation suite on the [email protected] computing platform to perform extensive molecular simulations and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. These simulations strongly implicated a conformational selection mechanism for Phe association with ACT domain dimers and revealed protein motions that act as a gating mechanism for Phe binding. The MSMs also illuminate a highly mobile hairpin loop, consistent with experimental findings also presented here that the PAH variant L72W does not shift the PAH structural equilibrium toward the activated state. Finally, simulations of ACT domain monomers are presented, in which spontaneous transitions between resting-state and activated conformations are observed, also consistent with a mechanism of conformational selection. These mechanistic details provide detailed insight into the regulation of PAH activation and provide testable hypotheses for the development of new allosteric effectors to correct structural and functional defects in PAH. Phenylalanine hydroxylase (PAH; 2The abbreviations used are: PAHphenylalanine hydroxylaseRS-PAHresting state phenylalanine hydroxylaseA-PAHactivated phenylalanine hydroxylasetICAtime-structure–based independent component analysisMSMMarkov state modelPKUphenylketonuriarPAHrat PAHTPTtransition path theoryIECion-exchange chromatographytICtICA componentSUMOsmall ubiquitin-like modifierBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propaneYTyeast extract–tryptone. EC 1.14.16.1) functions in humans to control free phenylalanine (Phe), an essential amino acid that is neurotoxic at elevated levels. Failure to control Phe, most often due to defects in PAH, results in hyperphenylalaninemia or phenylketonuria (PKU), which is the most common inborn error of amino acid metabolism. PAH catalyzes the conversion of Phe to tyrosine at the enzyme active site but also binds Phe at an allosteric site that sits at a subunit–subunit interface of a PAH tetramer. This intersubunit interface, which is present in activated PAH (A-PAH) and absent in resting-state PAH (RS-PAH), lies between ACT subdomains located diagonally across the tetramer (Fig. 1, a and b). ACT domains, which can serve in ligand sensing, were named for the first three proteins in which they were identified (1Aravind L. Koonin E.V. Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches.J. Mol. Biol. 1999; 287 (10222208): 1023-104010.1006/jmbi.1999.2653Crossref PubMed Scopus (370) Google Scholar). Phe stabilization of A-PAH controls the equilibrium between RS-PAH and A-PAH and thus allows Phe to regulate PAH activity (2Jaffe E.K. New protein structures provide an updated understanding of phenylketonuria.Mol. Genet. Metab. 2017; 121 (28645531): 289-29610.1016/j.ymgme.2017.06.005Crossref PubMed Scopus (22) Google Scholar). The Phe-stabilized conformational change is coupled to exposure of the enzyme active site (3Jaffe E.K. Stith L. Lawrence S.H. Andrake M. Dunbrack Jr., R.L. A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics.Arch. Biochem. Biophys. 2013; 530 (23296088): 73-8210.1016/j.abb.2012.12.017Crossref PubMed Scopus (49) Google Scholar), thus activating the enzyme. phenylalanine hydroxylase resting state phenylalanine hydroxylase activated phenylalanine hydroxylase time-structure–based independent component analysis Markov state model phenylketonuria rat PAH transition path theory ion-exchange chromatography tICA component small ubiquitin-like modifier 1,3-bis[tris(hydroxymethyl)methylamino]propane yeast extract–tryptone. The crystal structure of a Phe-bound ACT domain dimer from truncated human PAH (Protein Data Bank (PDB) code 5FII) (4Patel D. Kopec J. Fitzpatrick F. McCorvie T.J. Yue W.W. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.Sci. Rep. 2016; 6 (27049649)2374810.1038/srep23748Crossref PubMed Scopus (41) Google Scholar) shows key differences from a homology model of the putative ligand-free ACT domain dimer (3Jaffe E.K. Stith L. Lawrence S.H. Andrake M. Dunbrack Jr., R.L. A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics.Arch. Biochem. Biophys. 2013; 530 (23296088): 73-8210.1016/j.abb.2012.12.017Crossref PubMed Scopus (49) Google Scholar). The homology model was constructed using monomeric rat PAH (rPAH; see Fig. S1 for sequence comparison) ACT domain (PDB code 1PHZ) as a template with the dimeric form modeled by threading onto the ACT domain dimer from phosphoglycerate dehydrogenase (PDB code 1PSD). Unliganded monomeric ACT domains have identical conformations in most available crystal structures that represent RS-PAH (PDB codes 1PHZ, 2PHM, 5DEN, and 5FGJ) (5Kobe B. Jennings I.G. House C.M. Michell B.J. Goodwill K.E. Santarsiero B.D. Stevens R.C. Cotton R.G. Kemp B.E. Structural basis of autoregulation of phenylalanine hydroxylase.Nat. Struct. Biol. 1999; 6 (10331871): 442-44810.1038/8247Crossref PubMed Scopus (195) Google Scholar, 6Arturo E.C. Gupta K. Héroux A. Stith L. Cross P.J. Parker E.J. Loll P.J. Jaffe E.K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (26884182): 2394-239910.1073/pnas.1516967113Crossref PubMed Scopus (39) Google Scholar7Meisburger S.P. Taylor A.B. Khan C.A. Zhang S. Fitzpatrick P.F. Ando N. Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering.J. Am. Chem. Soc. 2016; 138 (27145334): 6506-651610.1021/jacs.6b01563Crossref PubMed Scopus (66) Google Scholar). This conformation differs from the Phe-bound crystal structure (PDB code 5FII) by having a helical turn at residues 61–64 (e.g. PDB code 5DEN), which, when overlaid on the Phe-bound ACT domain dimer structure, fills the cavity of the allosteric Phe-binding site. The homology model and the crystal structure also differ in their β-strand register at the dimer interface. Molecular simulations of the homology model and crystal structure, in monomeric and dimeric forms and in the presence and absence of Phe, offer valuable insights into the Phe binding mechanism not available by other methods. Whereas previous experimental studies have estimated binding equilibria for Phe to the preformed ACT domain dimer (8Zhang S. Roberts K.M. Fitzpatrick P.F. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.Biochemistry. 2014; 53 (25299136): 6625-662710.1021/bi501109sCrossref PubMed Scopus (26) Google Scholar), simulations can provide estimates of Phe binding pathways and rates crucial to understanding the molecular mechanism of allosteric activation (9Gu S. Silva D.-A. Meng L. Yue A. Huang X. Quantitatively characterizing the ligand binding mechanisms of choline binding protein using Markov state model analysis.PLoS Comput. Biol. 2014; 10 (25101697)e100376710.1371/journal.pcbi.1003767Crossref PubMed Scopus (55) Google Scholar10Huang X. De Fabritiis G. Understanding molecular recognition by kinetic network models constructed from molecular dynamics simulations.Adv. Exp. Med. Biol. 2014; 797 (24297279): 107-11410.1007/978-94-007-7606-7_9Crossref PubMed Scopus (2) Google Scholar, 11Stanley N. Pardo L. Fabritiis G.D. The pathway of ligand entry from the membrane bilayer to a lipid G protein-coupled receptor.Sci. Rep. 2016; 6 (26940769)2263910.1038/srep22639Crossref PubMed Scopus (64) Google Scholar, 12Paul F. Noé F. Weikl T.R. Identifying conformational-selection and induced-fit aspects in the binding-induced folding of PMI from Markov state modeling of atomistic simulations.J. Phys. Chem. B. 2018; 122 (29522679): 5649-565610.1021/acs.jpcb.7b12146Crossref PubMed Scopus (17) Google Scholar, 13Plattner N. Noé F. Protein conformational plasticity and complex ligand-binding kinetics explored by atomistic simulations and Markov models.Nat. Commun. 2015; 6 (26134632)765310.1038/ncomms8653Crossref PubMed Scopus (260) Google Scholar14Malmstrom R.D. Kornev A.P. Taylor S.S. Amaro R.E. Allostery through the computational microscope: cAMP activation of a canonical signalling domain.Nat. Commun. 2015; 6 (26145448)758810.1038/ncomms8588Crossref PubMed Scopus (67) Google Scholar). In particular, because the putative unliganded homology model represents an alternative dimer conformation where the monomers are very close to the RS-PAH conformation, ab initio binding simulations can distinguish between conformational selection versus induced fit ligand binding mechanisms. Moreover, the conformational motions seen in molecular simulations can be used to understand the effects of mutations and to generate testable hypotheses about alternative conformational states that could be targeted by allosteric effectors (15Bowman G.R. Bolin E.R. Hart K.M. Maguire B.C. Marqusee S. Discovery of multiple hidden allosteric sites by combining Markov state models and experiments.Proc. Natl. Acad. Sci. U.S.A. 2015; (201417811)Crossref Scopus (130) Google Scholar). Until now, only submicrosecond simulations of the ACT domain monomer in the absence of Phe have been performed (16Carluccio C. Fraternali F. Salvatore F. Fornili A. Zagari A. Structural features of the regulatory ACT domain of phenylalanine hydroxylase.PLoS One. 2013; 8 (24244510): e79413-e7948210.1371/journal.pone.0079482Crossref PubMed Scopus (17) Google Scholar). Here, ∼633 μs of trajectory data obtained from parallel explicit-solvent molecular dynamics simulations were performed and analyzed to elucidate 1) the conformational dynamics of the ACT domain monomer, 2) the conformational dynamics of Phe-binding encounter complexes of the ACT domain dimer, and 3) pathways and rates of Phe binding to ACT domain dimer conformations. As described below, Markov state model analysis of the trajectory data implicates a conformational selection mechanism for Phe association with ACT domain dimers and reveals key conformational motions coupled to Phe binding. One of these motions corresponds to a ligand gating mechanism, whereas another reflects mobility in a hairpin loop region. Experimental measurements on PAH with a bulky amino acid substitution in this region do not show a shift in the RS-PAH to A-PAH equilibrium, corroborating the role of flexibility in the hairpin loop. Multiple independent methods were used to estimate Phe binding rates to the preformed regulatory ACT domain dimer poses: 1) the distribution of observed binding times, 2) the numbers of binding events, 3) implied timescales from Markov state models, and 4) binding flux estimates from transition path theory (TPT). Bayesian inference was used to estimate the rate of free Phe binding to an ACT domain dimer from the distribution of binding times. Given N observed binding events, each with an observed time to binding ti, i = 1, …, N, each binding time ti was assumed to come from a Poisson distribution, P(t|k) = k exp(−kt), for some unknown binding rate, k. The likelihood of observing the data given the rate k is P(t1, t2, …, tN|k) = Πi P(ti|k) = kN exp(−k∑iti). By Bayes’ theorem, the posterior probability of the value of k, given the observed binding times ti, is P(k|t1, t2, …, tN) ∝ P(t1, t2, …, tN|k) P(k) where P(k) is a prior distribution. Two common choices were considered for the prior distribution: the uniform distribution (P(k) ∼ 1) and the noninformative Jeffreys prior (P(k) ∼ 1/k). Using the N = 29 observed binding times (Table S4), the maximum posterior probability yields an estimate of k = 6.28 × 107 s−1 m−1 (95% confidence interval, 4.63–8.50 × 107 s−1 m−1) for uniform prior and k = 6.03 × 107 s−1 m−1 (95% confidence interval, 4.65–8.54 × 107 s−1 m−1) using a Jeffreys prior (Fig. S10). As described in Shirts and Pande (17Shirts M.R. Pande V.S. Mathematical analysis of coupled parallel simulations.Phys. Rev. Lett. 2001; 86 (11384401): 4983-498710.1103/PhysRevLett.86.4983Crossref PubMed Scopus (86) Google Scholar), simulating M parallel trajectories of a Poisson process results in a probability of observing a binding event after time t is given by PM(t) = Mk exp(−Mkt). Therefore, for a collection of trajectories of different lengths, the expected number of binding events 〈n〉 is 〈n〉(k) = ∫M(t)k exp(−M(t)kt)dt where M(t) is the number of trajectories that reach a length of t (18Voelz V.A. Bowman G.R. Beauchamp K. Pande V.S. Molecular simulation of ab initio protein folding for a millisecond folder NTL9(1–39).J. Am. Chem. Soc. 2010; 132 (20070076): 1526-152810.1021/ja9090353Crossref PubMed Scopus (409) Google Scholar). Because the analysis suggests that only crystal-like ACT domain dimer poses are competent for binding (see Fig. 1g), a subset of trajectory data corresponding to crystal-like dimer poses was analyzed (starting conformations 0–9; Fig. S11). The 29 binding events that were observed (assuming ±5.2 from binomial finite sampling error) corresponds to rates in the range of 2–6 × 107 s−1 m−1 (Fig. S12). The slowest MSM implied timescale, τ1 = 256 ± 65 ns, corresponds to Phe binding (see Fig. 2 and Fig. S6). The observed relaxation rate corresponding to two-state binding is therefore kobs = kon + koff = 1/cτ1 where c = 99.52 mm is the effective concentration of Phe in the simulations. Because koff ≪ kon, the estimated binding rate is kon = 1/cτ1 = 3.9 × 107 s−1 m−1. A bootstrap estimate of standard error in (ln τ1) yields upper and lower estimates of 5.1 and 3.0 × 107 s−1 m−1, respectively. Of the 480 trajectories generated, 29 independent Phe binding events were observed (Movie S1). Binding was monitored using the average distance between the Phe ligand and three residues that show close contact to the Phe ligands in the Phe-bound ACT domain dimer crystal structure: Leu48, Leu62, and Ile65 (Fig. 1c). From the distribution of observed binding times (Table S4), a binding rate of 6 × 107 s−1 m−1 to the ACT domain dimer is inferred (Fig. 1d). This value is corroborated by estimates from the number of binding events and MSM-based rate estimates (Table 1).Table 1Estimates of binding rates of free Phe to the preformed ACT domain dimerMethodRateaThe average of all methods is 5 ± 3 × 107 s−1 m−1.Uncertainty (lower–upper bound)(× 107 s−1 m−1)Binding time distributionUniform prior6.284.63–8.50Jeffreys prior6.034.65–8.54Numbers of binding events4.02.0–6.0MSM implied timescales3.93.0–5 .1Transition path theory6.01.0–6 .3a The average of all methods is 5 ± 3 × 107 s−1 m−1. Open table in a new tab Physiologically, the estimated binding rate is slower than the theoretical diffusion limit (∼109 s−1 m−1) in the range typical for small molecules binding to protein targets (19Pang X. Zhou H.-X. Rate constants and mechanisms of protein-ligand binding.Annu. Rev. Biophys. 2017; 46 (28375732): 105-13010.1146/annurev-biophys-070816-033639Crossref PubMed Scopus (43) Google Scholar, 20Kokh D.B. Amaral M. Bomke J. Grädler U. Musil D. Buchstaller H.-P. Dreyer M.K. Frech M. Lowinski M. Vallee F. Bianciotto M. Rak A. Wade R.C. Estimation of drug-target residence times by τ-random acceleration molecular dynamics simulations.J. Chem. Theory Comput. 2018; 14 (29768913): 3859-386910.1021/acs.jctc.8b00230Crossref PubMed Scopus (112) Google Scholar). For comparison, the experimentally measured binding rate of benzamidine (similar in size to Phe) to trypsin is ∼2.9 × 107 s−1 m−1 (21Guillain F. Thusius D. Use of proflavine as an indicator in temperature-jump studies of the binding of a competitive inhibitor to trypsin.J. Am. Chem. Soc. 1970; 92 (5449454): 5534-553610.1021/ja00721a051Crossref PubMed Scopus (69) Google Scholar). It should be kept in mind that our rate estimate reflects the binding rate to the preformed ACT domain dimer and that ACT domain dimerization would likely limit the rate of PAH activation at high Phe concentrations. Sample binding traces are shown in Fig. 1, e and f. Of the 29 binding events, all but one are found in trajectories started from poses resembling the liganded crystal structure (Fig. 1g). The lone exception is found in a trajectory for which an unliganded homology model–like dimer pose first transitions to a more crystal structure–like pose before Phe binds (Fig. 1, f and h, orange trace). This dimer pose may still be suboptimal for binding because unbinding is later observed. No unbinding events are observed in the 28 other binding trajectories or in any of the simulations starting from the Phe-bound crystal structure (PDB code 5FII). The absence of simulated unbinding events is consistent with estimates of unbinding rates. Zhang et al. (8Zhang S. Roberts K.M. Fitzpatrick P.F. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.Biochemistry. 2014; 53 (25299136): 6625-662710.1021/bi501109sCrossref PubMed Scopus (26) Google Scholar) have used analytic ultracentrifugation to estimate a dissociation constant (Kd) of 8.3 μm for each Phe molecule’s association to an ACT domain dimer. Using our estimated kon value, this implies a Phe dissociation rate koff = kon/Kd of about 500 s−1 and would suggest that millisecond-timescale trajectories would be required to observe unbinding, whereas our longest trajectories are around 1 μs. To visualize the conformational dynamics of the collection of ACT domain dimer poses seen in the ab initio binding trajectories, time-structure–based independent component analysis (tICA) was performed to project trajectory coordinates to a low-dimensional subspace representing the slowest motions of the dimers. The largest tICA component (tIC1) shows slow interconversion between crystal structure–like and homology model–like dimer poses, whereas the next largest component (tIC2) shows motions corresponding to ACT domain dimer dissociation (Fig. 1h). Dimer dissociation is not seen for crystal structure–like poses. Despite the numerous interactions between free Phe and the ACT domain dimer poses in the simulations, both ligand binding and dimer association are found to be highly dependent on the dimer pose. This finding suggests a conformational selection mechanism of Phe binding whereby formation of a binding-competent dimer is required before association of Phe; in other words, the formation of the bound dimer is not a Phe-induced conformational change. Simulations of an ACT domain dimer in high concentrations of Phe additionally provide a detailed picture of Phe-binding hot spots on the dimer surface other than the allosteric Phe-binding site. Significant binding propensity between free Phe and Phe80 (Fig. S13) is observed, which is intriguing because a somewhat buried Phe80 of RS-PAH participates in stabilizing that subunit conformation through cation–π interactions with arginine residues on both the catalytic and multimerization domains on the same subunit (6Arturo E.C. Gupta K. Héroux A. Stith L. Cross P.J. Parker E.J. Loll P.J. Jaffe E.K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (26884182): 2394-239910.1073/pnas.1516967113Crossref PubMed Scopus (39) Google Scholar). Further analysis, however, suggests that the observed binding propensity arises mainly from the high concentration of free Phe used in the simulations; on- and off-rates suggest low overall affinity between free Phe and Phe80 of the ACT domain dimer (Kd ∼ 200 mm; see supporting methods, Fig. S14, and Table S5). Note that Phe80, which is on the eight-stranded β-sheet of the ACT domain dimer, is predicted to be facing inward toward the C-terminal four-helix bundle of the A-PAH tetramer model (refer to Fig. 1b). Simulations of the ACT dimer poses in the absence of Phe show dimer dissociation for homology-like dimer poses but not for crystal structure–like poses (Fig. 1h). This finding is consistent with significant in vitro data on the isolated PAH ACT domain that suggest spontaneous dimer formation in the absence of Phe and the stabilization of this dimer in the presence of Phe (8Zhang S. Roberts K.M. Fitzpatrick P.F. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.Biochemistry. 2014; 53 (25299136): 6625-662710.1021/bi501109sCrossref PubMed Scopus (26) Google Scholar, 22Zhang S. Fitzpatrick P.F. Identification of the allosteric site for phenylalanine in rat phenylalanine hydroxylase.J. Biol. Chem. 2016; 291 (26823465): 7418-742510.1074/jbc.M115.709998Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 23Li J. Ilangovan U. Daubner S.C. Hinck A.P. Fitzpatrick P.F. Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase.Arch. Biochem. Biophys. 2011; 505 (20951114): 250-25510.1016/j.abb.2010.10.009Crossref PubMed Scopus (34) Google Scholar). Other studies show that although full-length RS-PAH in the absence of Phe samples both tetramer and dimer, the addition of Phe favors a stable tetramer (presumably A-PAH) (3Jaffe E.K. Stith L. Lawrence S.H. Andrake M. Dunbrack Jr., R.L. A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics.Arch. Biochem. Biophys. 2013; 530 (23296088): 73-8210.1016/j.abb.2012.12.017Crossref PubMed Scopus (49) Google Scholar). To elucidate the mechanism of Phe binding to the ACT domain dimer, MSMs of the conformational dynamics associated with binding using the coordinates of Phe ligands and the protein residues surrounding a single binding site were constructed (see “Experimental procedures” and supporting information). Before doing this, however, the possibility of any Phe-binding cooperativity was probed by building MSMs of both binding sites in the Phe-bound dimer (PDB code 5FII). The results suggest that conformational dynamics in each binding site are independent of each other (data not shown). Binding statistics also support noncooperative Phe binding to the preformed dimer: of the 29 binding events observed in 480 trajectories, two of those 29 trajectories show double Phe binding events (Fig. S15), consistent with a ∼6% independent probability of observing binding for each site (29/480 ≈ 2/29). Consistent with this result are experimental measurements of Phe-dependent ACT domain dimerization in which the model that best fit sedimentation data was the one with identical and independent Phe binding to the preformed ACT domain dimer (8Zhang S. Roberts K.M. Fitzpatrick P.F. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.Biochemistry. 2014; 53 (25299136): 6625-662710.1021/bi501109sCrossref PubMed Scopus (26) Google Scholar) as well as isothermal titration calorimetry studies of the truncated ACT dimer (7Meisburger S.P. Taylor A.B. Khan C.A. Zhang S. Fitzpatrick P.F. Ando N. Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering.J. Am. Chem. Soc. 2016; 138 (27145334): 6506-651610.1021/jacs.6b01563Crossref PubMed Scopus (66) Google Scholar). We then proceeded to construct an MSM of 75 metastable conformational states for Phe ligands and protein residues surrounding a single binding site. The results reveal structural intermediates along the Phe binding pathway coupled to protein dynamics (Fig. 2). The tICA projections show that the slowest-timescale motions are coupled to binding events with the most significant changes in interresidue distances corresponding to residues that gate the entry of Phe (Glu44/Val45 and Asp59/Val60/Asn61) (Fig. 2, blue). Strikingly, the MSM shows that the protein loop containing Val45 must swing open to allow access to the binding site. In all binding trajectories, this gate must open before binding can occur. Once the ligand is bound, the gate closes, helping to stabilize the bound state. More details describing this motion can be found in Table S6 and Figs. S16 and S17. In the absence of free Phe, the slowest two motions of the dimer are also found to occur within the same area that functions as the binding gate for Phe binding (Table S7 and Fig. S18). This suggests that this intrinsic motion is independent of Phe concentration, again consistent with conformational selection. The next-slowest conformational motion corresponds to bending of the hairpin loop containing Leu72 (Fig. 2, magenta). Although this motion occurs on a timescale (∼100 ns) similar to that of binding, it does not appear coupled to binding; binding occurs regardless of whether the hairpin loop is bent. Using transition path theory, it is estimated that about 8% of binding flux occurs through bent-hairpin pathways (Fig. S8). The mobility of this hairpin loop helps explain a number of experimental observations. First, in the crystal structure of the Phe-bound ACT domain dimer (PDB code 5FII), Leu72 is unresolved in one of the four chains, indicative of high mobility. Second, hydrogen/deuterium exchange studies on full-length rPAH (25Li J. Dangott L.J. Fitzpatrick P.F. Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry.Biochemistry. 2010; 49 (20307070): 3327-333510.1021/bi1001294Crossref PubMed Scopus (34) Google Scholar) showed that a peptide containing residues 67–81, which includes a significant portion of the β-strands on either side of the hairpin loop containing Leu72, displays modest hydrogen/deuterium exchange. There is no difference in hydrogen/deuterium exchange for the peptide in the absence or presence of 5 mm Phe, which is consistent with the prediction from the current molecular dynamics simulations that the hairpin loop’s mobility is inherent to the structure and not dependent on Phe binding. To investigate the mobility of the hairpin loop region, the L72W variant of the rat protein was prepared and tested for the robustness of the RS-PAH ⇔ A-PAH structural equilibrium. In the full-length crystal structure of rat RS-PAH (6Arturo E.C. Gupta K. Héroux A. Stith L. Cross P.J. Parker E.J. Loll P.J. Jaffe E.K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (26884182): 2394-239910.1073/pnas.1516967113Crossref PubMed Scopus (39) Google Scholar), the hairpin loop containing Leu72 is located between the multimerization domain and a neighboring catalytic subunit. This is in contrast to an early composite homology model of RS-PAH, made by combining two two-domain PAH structures, both containing the catalytic domain, which suggested a potential clash between Leu72 of the ACT domain and Ile432 in the multimerization domain (6Arturo E.C. Gupta K. Héroux A. Stith L. Cross P.J. Parker E.J. Loll P.J. Jaffe E.K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (26884182): 2394-239910.1073/pnas.1516967113Crossref PubMed Scopus (39) Google Scholar, 26Erlandsen H. Stevens R.C. The structural basis of phenylketonuria.Mol. Genet. Metab. 1999; 68 (10527663): 103-12510.1006/mgme.1999.2922Crossref PubMed Scopus (126) Google Scholar). The RS-PAH ⇔ A-PAH structural equilibrium was characterized via measurements of the enzyme’s kinetics, intrinsic Trp fluorescence, and affinity for an ion-exchange resin as described below. Taken together, these data are uniformly consistent wi" @default.
• W2895679935 created "2018-10-12" @default.
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• W2895679935 title "Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding" @default.
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