Wrangling Shape-Shifting Morpheeins to Tackle Disease and Approach Drug Discovery.

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.

Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.

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.

Porphobilinogen synthase: An equilibrium of different assemblies in human health.

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.

Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium.

Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.

Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding.

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.

New protein structures provide an updated understanding of phenylketonuria.

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.

The Remarkable Character of Porphobilinogen Synthase.

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; a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb.

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].

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

Background: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain.

wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum.

Impact of quaternary structure dynamics on allosteric drug discovery.

The morpheein model of allosteric regulation draws attention to proteins that can exist as an equilibrium of functionally distinct assemblies where: one subunit conformation assembles into one multimer; a different subunit conformation assembles into a different multimer; and the various multimers are in a dynamic equilibrium whose position can be modulated by ligands that bind to a multimer-specific ligand binding site. The case study of porphobilinogen synthase (PBGS) illustrates how such an equilibrium holds lessons for disease mechanisms, drug discovery, understanding drug side effects, and identifying proteins wherein drug discovery efforts might focus on quaternary structure dynamics. The morpheein model of allostery has been proposed as applicable for a wide assortment of disease-associated proteins (Selwood, T.

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.

Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (PBGS, a.k.

Facile Synthesis of Mutagen X (MX): 3-Chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one.

3-Chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one (Mutagen X, MX) was synthesized in six steps from commercially-available and inexpensive starting materials (27% overall yield). This synthesis enables the preparation of MX analogs and does not require the use of chlorine gas, as do previously reported methods.

Dynamic dissociating homo-oligomers and the control of protein function.

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands.

The morpheein model of allostery: evaluating proteins as potential morpheeins.

An equilibrium mixture of alternate quaternary structure assemblies can form a basis for allostery. The morpheein model of allostery is a concerted dissociative model that describes an equilibrium of alternate quaternary structure assemblies whose architectures are dictated by alternate conformations in the dissociated state. Kinetic and biophysical anomalies that suggest that the morpheein model of allostery applies for a given protein of interest are briefly described.

Allostery and the dynamic oligomerization of porphobilinogen synthase.

The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer∗⇔ 6mer∗. The "∗" represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable.

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.

Diverse clinical compounds alter the quaternary structure and inhibit the activity of an essential enzyme.

An in vitro evaluation of the Johns Hopkins Clinical Compound Library demonstrates that certain drugs can alter the quaternary structure of an essential human protein. Human porphobilinogen synthase (HsPBGS) is an essential enzyme involved in heme biosynthesis; it exists as an equilibrium of high-activity octamers, low-activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. Decreased HsPBGS activity is implicated in toxicities associated with lead poisoning and 5-aminolevulinate dehydratase (ALAD) porphyria, the latter of which involves hexamer-favoring HsPBGS variants.

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation.

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen.

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.

Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin.

MORPHEEINS - A NEW PATHWAY FOR ALLOSTERIC DRUG DISCOVERY.

The morpheein model of allosteric regulation can be applied as a novel approach to the discovery of small molecule allosteric modulators of protein function. Morpheeins are homo-oligomeric proteins where, under physiological conditions, the oligomer can dissociate, the dissociated units can change conformation, and the altered conformational state can reassociate to a structurally and functionally distinct oligomer. This phenomenon serves as a basis for allostery, as a basis for conformational diseases, as a basis for drug discovery, and may be applicable to personalized medicine such as in the prediction of drug side effects.

Allosteric inhibition of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis.

Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation.

The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al.