Enzymes that regulate their activity by modulating an equilibrium of alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) constitute a recently described mode of allostery. The oligomeric equilibrium for porphobilinogen synthase (PBGS) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. A phylogenetically diverse allosteric site specific to hexamers is proposed as an inhibitor binding site. Inhibitor binding is predicted to draw the oligomeric equilibrium toward the low-activity hexamer. In silico docking enriched a selection from a small-molecule library for compounds predicted to bind to this allosteric site. In vitro testing of selected compounds identified one compound whose inhibition mechanism is species-specific conversion of PBGS octamers to hexamers. We propose that this strategy for inhibitor discovery can be applied to other proteins that use the morpheein model for allosteric regulation.

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 ϳ111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventyseven 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.Human porphobilinogen synthase (PBGS, 2 EC 4.2.1.24, also known as 5-aminolevulinate dehydratase) exists as a quaternary structure equilibrium consisting of a high activity octamer, a low activity hexamer, and a dimer that can take on two distinct conformations, each of which dictates assembly to either the octamer or the hexamer (see Fig. 1a) (1). For the wild type protein at neutral pH, the octamer is the dominant assembly in the equilibrium (1). As PBGS catalyzes the first common step in the biosynthesis of the tetrapyrroles such as heme, low activity mutations in the human population are associated with the disease ALAD porphyria (2); all eight porphyria-associated PBGS mutations increase the propensity of the human protein to exist in the low activity hexameric assembly, thus establishing the physiologic relevance of the quaternary structure equilibrium to human health (3). In addition to ALAD porphyria, PBGS inhibition by divalent lead is a primary consequence of lead poisoning. Factors that stabilize the hexamer will further inhibit PBGS activity and, thus, potentiate the physiologic effects of lead poisoning.The arrangement of the subunits in the PBGS hexamer creates a surface cavity that is not present in the octamer or the dimers (see Fig. 1b) (4). Ligand binding to this cavi...

The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.

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. The PBGS family exhibits a complex use of metal ions (Zn 2؉ and Mg 2؉ ) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ϳ320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7-8.5), with a K m for aminolevulinic acid of ϳ150 M and specific activity of ϳ24 mol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg 2؉ but not Zn 2؉ and shows two Mg 2؉ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg 2؉ -binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg 2+ GDP reveals two new binding conformations-in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the γ-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDPbound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.