First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

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 Å 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 Phe-stabilized 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.

A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics.

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ∼90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the intersubunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic.

Allosteric inhibition of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

Docking to large allosteric binding sites on protein surfaces.

The inactive porphobilinogen synthase (PBGS) hexamer has an oligomer-specific and phylogenetically variable surface cavity that is not present in the active octamer. The octamer and hexamer are components of a dynamic quaternary structure equilibrium characteristic of morpheeins. Small molecules that bind to the hexamer-specific surface cavity, which is at the interface of three subunits, are predicted to act as allosteric inhibitors that function by drawing the oligomeric equilibrium toward the hexamer. We used GLIDE as a tool to enrich a 250,000 molecule library for molecules with enhanced probability of acting as hexamer-stabilizing allosteric inhibitors of PBGS from Yersinia enterocolitica. Eighty-six compounds were tested in vitro and five showed hexamer stabilization. We discuss the application of computational docking to surface cavities as an approach to find allosteric modulators of protein function with specific reference to morpheeins that function as an equilibrium of non-additive quaternary structure assemblies.

Pseudomonas aeruginosa porphobilinogen synthase assembly state regulators: hit discovery and initial SAR studies.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of the essential heme, chlorophyll and vitamin B(12) heme pigments. PBGS activity is regulated by assembly state, with certain oligomers exhibiting biological activity and others either partially or completely inactive, affording an innovative means of allosteric drug action. Pseudomonas aeruginosa PBGS is functionally active as an octamer, and inactive as a dimer. We have identified a series of compounds that stabilize the inactive P. aeruginosa dimer by a computational prescreen followed by native PAGE gel mobility shift analysis. From those results, we have prepared related thiadiazoles and evaluated their ability to regulate P. aeruginosa PBGS assembly state.

ALAD porphyria is a conformational disease.

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins).

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.

Substrate-induced interconversion of protein quaternary structure isoforms.

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.

A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase.

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.