Phenylalanine hydroxylase (PAH, EC 1.14.16.1) is an enzyme that converts L-phenylalanine (Phe) to L-tyrosine, regulating normal Phe blood levels (~50 - 120 µM). In phenylketonuria (PKU), inherited defects in PAH result in hyperphenylalaninemia where Phe blood levels can become neurotoxic (up to ~2.5 mM). Recent atomic structures of mammalian PAH combined with solution scattering analyses have lent insight into the allosterically regulated conformati onal changes that occur between a resting state tetramer to a highly active form in rising Phe concentrations. This transition occurs via a conformational selection model, where the binding of allosteric Phe at a distal site within the regulatory (ACT) domain modulates this facile equilibrium. Our recent study (Arturo et. al., Biochimie 2021) shows that disrupting a single cation-π interaction between the regulatory and catalytic domains (Phe80 variants) results in PAH variants with unique activity and biochemical profiles. In this study, we used a combination of analytical ultracentrifugation (AUC) and size-exclusion chromatography with synchrotron X -ray scattering (SEC-SAXS) to investigate the oligomerization and solution conformations of these variants. By modeling the SEC-SAXS data with atomistic models, we gained insights into the arrangement of these intermediates in different functional forms. Our work reveals the presence of various oligomeric assemblies and conformers in solution, and insights into the modulation of PAH activity by its

Homo-multimeric proteins that can come apart, change shape, and reassemble differently with functional consequences have been called morpheeins and/or transformers; these provide a largely unexplored context for understanding disease and developing allosteric therapeutics. This article describes such proteins within the context of protein structure dynamics, provides one detailed example related to an inborn error of metabolism and potential herbicide development, and describes the context for applying these ideas for understanding disease and designing bioactive molecules, such as therapeutics.

Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.

Herein we describe progress in a class of shape‐shifting proteins originally called morpheeins. These are homomultimers that can dissociate, change conformation in the dissociated state, and reassemble into a structurally and functionally distinct multimer. The original observation was of an octamer/dimer/dimer*/hexamer equilibrium wherein the conformational change was a hinge motion between folded domains of porphobilinogen synthase (PBGS). This phenomenon is established as a mechanism for allosteric regulation, druggable, and the structural basis for an inborn error of metabolism.Many aspects of the original observations in the early 2000s were unexpected and peculiar; suggesting a structural metastability. At the time, vocabulary to describe such multi‐structural and multi‐functional protein dynamics were limited. We defined morpheeins as (homo‐multimeric) proteins that can come apart, change shape and reassemble differently with functional consequences. A Wikipedia page introduced a dice model of a morpheein (see image), and showed how multimer‐specific surface cavities can provide a general mechanism for drug action. In the beginning, multimeric proteins with more than one shape (in the absence of refolding) strongly defied the one sequence, one structure, one function paradigm. Two decades later, there are a growing number of examples of multimeric proteins with multiple structures and other proteins with multiple functions, many in the absence of any chemical modification or significant refolding.The quaternary structure dynamics of PBGS have now been extensively described. Interestingly, one consequence of our focus on PBGS is that the literature began equated the specific properties of PBGS with all of the factors that must be in place for a protein to be classified as a morpheein. This is akin to saying that in order to be considered a mammal, an organism must have all of the characteristics of a rat. Herein, we set straight the definition of a morpheein; it is a protein that can come apart, change conformation in the dissociated state, and reassemble to a structurally and functionally distinct multimer. Alternate assemblies need not have different stoichiometries. Assembly interchange need not be triggered by ligand binding or catalysis. Functional difference need not be high activity and low activity (as in allosteric regulation of enzyme action), but can be moonlighting activities. Often the conformational change is a repositioning of folded domains relative to each other.An excellent example of a moonlighting protein that appears to be a morpheein, and has also been called a transformer, is the VP40 protein of the Ebola virus. It too exists as dimers or hexamer or octamer and each alternate assembly is responsible for an alternate function in the viral life cycle. The individual assemblies are stabilized through interaction with a “third party”. A third likely example is the family of alternate assemblies of ribonucleotide reductase, whose timely interchange amongst alternate assemblies appears essential to the regulation of nucleotide pools. A fourth example is HIV integrase, whose three domains reposition relative to each other as part of viral integration. These examples will be illustrated.Support or Funding InformationNIH grants 5R01 NS100081 and P30 CA006927Dice image depicting an interchange of morpheein forms and a ligand that stabilizes one form.Figure 1

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria (PKU). PAH equilibrates among long‐lived conformations, including resting‐state (RS‐PAH) and activated (A‐PAH), whose equilibrium position depends upon allosteric Phe binding to the A‐PAH conformation. The RS‐PAH conformation contains a stabilizing cation‐pi sandwich between Phe80, Arg123, and Arg240 (PDB entry 5DEN), which cannot exist in the A‐PAH conformation. Intrinsic protein fluorescence, enzyme kinetic analysis, native PAGE, size exclusion chromatography, limited proteolysis, and behavior on ion exchange resin are reported for F80A, F80D, F80L, and F80R, many as a function of [Phe]. These data indicate that amino acid substitutions at Phe80 destabilizes both the RS‐PAH and A‐PAH conformations so that intermediate, on‐pathway conformations are longer lived. The addition of Phe allows stabilization of the A‐PAH conformation. Kinetic characterization of F80A and F80D reflects allosteric activation while F80L and F80R are constitutively active. The reaction rates of all Phe80 variants suggest relief of a rate determining conformational change present in the wild type protein. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a central C‐terminal 4‐helix bundle, reflecting dynamic dissociation of the PAH tetramer. Under these conditions, the Phe80 variants show proteolytic hypersensitity in a linker region that repositions in the RS‐PAH to A‐PAH conformational interchange; this is protected by addition of Phe. We conclude that manipulation of Phe80 dramatically affects the conformational space sampled by PAH, increasing the population of intermediates between RS‐PAH and A‐PAH.Support or Funding InformationNIH 5R01‐NS100081; NIH P30 CA006927

Porphobilinogen synthase (PBGS) is an essential enzyme that catalyzes an early step in heme biosynthesis. An unexpected human PBGS quaternary structure dynamic drove the definition of morpheeins, which are protein multimers that dissociate, change shape, and re-assemble differently with functional consequences. Each PBGS monomer has two domains that can reposition through a hinge motion. Human PBGS exists in an equilibrium among high activity octamer, low activity hexamer, and low mole-fraction dimer in which the hinge motion occurs. The dimer conformation dictates the multimer architecture. An octamer-specific inter-subunit interaction responds to pH, resulting in a pH-dependence to the octamer-hexamer equilibrium. An inborn error of metabolism, ALAD porphyria, is caused by single amino acid substitutions that stabilize the hexamer relative to octamer. Drugs that stabilize the PBGS hexamer result in a drug side effect that can exacerbate porphyria. PBGS is essential for all organisms that require respiration, photosynthesis, or methanogenesis. Consequently, phylogenetic variation in PBGS multimerization equilibria provides insight into how Nature has harnessed oligomeric variation in the control of protein function. The dynamic multimerization of PBGS revealed the morpheein mechanism for allostery, a structural basis for inborn errors of metabolism, a quaternary structure focus for drug discovery and/or drug side effects, and a pathway toward new antibiotics or herbicides. The fortuitous discovery of PBGS quaternary structure dynamics arose from characterization of a low-activity single amino acid variant that dramatically stabilized the hexamer, whose existence had previously gone unnoticed.

Increasing numbers of proteins are being revealed to “shape shift” in physiologically relevant ways that do not dramatically alter secondary structure. As each has been discovered they have been called morpheeins, transformers, metamorphic polymorphs, and others. In each case the alternate assemblies have different functions ranging from “on vs. off” to “moonlighting”. These examples continue to surprise their discoverers because they defy the “one sequence, one structure, one function” paradigm in an unexpected way. These alternate assemblies are likely physiologically relevant as their interconversion is thermodynamically feasible and the equilibrium of alternate assemblies is responsive to biological signals (e.g. pH, ligands). Experience now teaches that single amino acid substitutions can also dramatically shift an equilibrium of alternate assemblies. In some cases this shift is reasonably hypothesized, or established, to contribute to diseases such as inborn errors of metabolism and cancer. We present examples culled both from our work and the literature of such shape‐shifting proteins whose equilibria respond to single amino acid substitutions. Prime examples come from diverse branches of life, including viral proteins, bacterial proteins, and human metabolic enzymes. The growing number of such examples suggests that alternate assemblies, which have been called the fifth level of protein structure, may be a common factor in protein structure‐function relationships. Experience teaches that this fifth level of structure, for a given protein, may be revealed by the unexpected biophysical behavior of single amino acid variants. Support or Funding Information NIH R01 NS100081 (EKJ)NIH R01 AI118016 (EOS) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.1) is the primary cause of phenylketonuria, the most common inborn error of amino acid metabolism. The dynamic domain rearrangements of this multimeric protein have thwarted structural study of the full-length form for decades, until now. In this study, a tractable C29S variant of hPAH (C29S) yielded a 3.06 angstrom resolution crystal structure of the tetrameric resting-state conformation. We used size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) to analyze the full-length hPAH solution structure both in the presence and absence of Phe, which serves as both substrate and allosteric activators. Allosteric Phe binding favors accumulation of an activated PAH tetramer conformation, which is biophysically distinct in solution. Protein characterization with enzyme kinetics and intrinsic fluorescence revealed that the C29S variant and hPAH are otherwise equivalent in their response to Phe, further supported by their behavior on various chromatography resins and by analytical ultracentrifugation. Modeling of resting-state and activated forms of C29S against SAXS data with available structural data created and evaluated several new models for the transition between the architecturally distinct conformations of PAH and highlighted unique intra- and inter-subunit interactions. Three best-fitting alternative models all placed the allosteric Phe-binding module 8-10 angstrom farther from the tetramer center than do all previous models. The structural insights into allosteric activation of hPAH reported here may help inform ongoing efforts to treat phenylketonuria with novel therapeutic approaches.

Phenylalanine hydroxylase (PAH) is an enzyme that catalyzes the conversion of phenylalanine to tyrosine, and functions in humans to control free phenylalanine (Phe), an essential amino acid that is neurotoxic at elevated levels. Mutations in PAH can result in phenylketonuria, which is the most common inborn error of amino acid metabolism. The transition from resting-state PAH to activated PAH requires formation of a new intersubunit interface that can be stabilized by Phe binding in an allosteric manner; formation of the new protein:protein interface is coupled to exposure of the active site, thus activating the enzyme. A recent crystal structure of this interface bound to Phe enables new insight into phenylalanine binding mechanism from molecular dynamics (MD) simulations. We performed massively parallel explicit-solvent simulations on the [email protected] distributed computing platform to elucidate pathways and rates of binding of Phe to the interface, which is comprised of a dimer of ACT domains. Time-lagged independent component analysis (tICA) of binding trajectories suggest a conformational selection mechanism. Markov state models (MSMs) constructed from the trajectory data reveal a key loop motion which acts as a “gatekeeper” of allosteric ligand binding. Binding rates estimated by different methods (MSMs, Transition Path Theory and Bayesian inference) agree well with each other. These results warrant further MD studies of the conformational dynamics involved in regulation of PAH activity, the effect of disease-associated mutations, and suggest future directions in simulation-based drug discovery.

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 Folding@home 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.

Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrrole pigments, which are essential for respiration, photosynthesis, and methanogenesis.A rare inborn error of metabolism, ALAD porphyria, is caused by human PBGS dysfunction.PBGS can participate in an equilibrium of architecturally distinct homomeric assemblies.An octamer contains intersubunit interactions that support high activity by allowing proper gating of active site access.A hexamer does not have these interactions and activity is low.The equilibrium between octamer and hexamer is governed by pH, and in some species by an allosteric activator that binds to an octamer-specific site.In the case of human PBGS, the ratio of octamer to hexamer is very sensitive to the protein sequence.Thus, we designed single amino acid variants that dramatically alter the human PBGS quaternary structure equilibrium to allow direct investigation of the interchange of structural isoforms.Of consequence to inborn errors of metabolism, all eight ALAD porphyria-associated variants favor the hexamer relative to wild type human PBGS.These variants occur throughout the sequence (F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M); octamer destabilization can be rationalized only for three of the eight variants (C132R, G133R, and R240W).Inexplicably, homomeric F12L assembles only to hexamer, though it can form a heteromeric octamer with wild-type PBGS.Had it not been for the stabile hexameric assembly of F12L, it is unlikely that we would have obtained a crystal structure for the PBGS hexamer, whose structure could not have been predicted from first principals nor homology.

Phenylketonuria (PKU) and less severe hyperphenylalaninemia (HPA) constitute the most common inborn error of amino acid metabolism, and is most often caused by defects in phenylalanine hydroxylase (PAH) function resulting in accumulation of Phe to neurotoxic levels. Despite the success of dietary intervention in preventing permanent neurological damage, individuals living with PKU clamor for additional non-dietary therapies. The bulk of disease-associated mutations are PAH missense variants, which occur throughout the entire 452 amino acid human PAH protein. While some disease-associated mutations affect protein structure (e.g. truncations) and others encode catalytically dead variants, most have been viewed as defective in protein folding/stability. Here we refine this view to address how PKU-associated missense variants can perturb the equilibrium among alternate native PAH structures (resting-state PAH and activated PAH), thus shifting the tipping point of this equilibrium to a neurotoxic Phe concentration. This refined view of PKU introduces opportunities for the design or discovery of therapeutic pharmacological chaperones that can help restore the tipping point to healthy Phe levels and how such a therapeutic might work with or without the inhibitory pharmacological chaperone BH4. Dysregulation of an equilibrium of architecturally distinct native PAH structures departs from the concept of “misfolding”, provides an updated understanding of PKU, and presents an enhanced foundation for understanding genotype/phenotype relationships.

Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do not use the allosteric Mg(2+), there is a spatially equivalent arginine-derived guanidium group. Deprotonation of this residue promotes formation of the hexamer and accounts for the basic arm of the bell-shaped pH vs activity profile of human PBGS. A human inborn error of metabolism known as ALAD porphyria is attributed to PBGS variants that favor the hexameric assembly. The existence of one such variant, F12L, which dramatically stabilizes the human PBGS hexamer, allowed crystal structure determination for the hexamer. Without this crystal structure and octameric PBGS structures containing the allosteric Mg(2+), it would have been difficult to decipher the structural basis for PBGS allostery. The requirement for multimer dissociation as an intermediate step in PBGS allostery was established by monitoring subunit disproportionation during the turnover-dependent transition of heteromeric PBGS (comprised of human wild type and F12L) from hexamer to octamer. One outcome of these studies was the definition of the dissociative morpheein model of protein allostery. The phylogenetically variable time scales for PBGS multimer interconversion result in atypical kinetic and biophysical behaviors. These behaviors can serve to identify other proteins that use the morpheein model of protein allostery.

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2): 73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 angstrom crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.