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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. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH; the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer* relative to wild type. The 6mer* has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer*. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects.
Porphobilinogen synthase (PBGS) is an ancient metabolic enzyme responsible for the biosynthesis of the fundamental monopyrrole that is the building block for myriad tetrapyrrolic cofactors required for life (porphyrin, chlorophyll, vitamin B12, siroheme, phytobilin, cofactor F430, etc.) . The PBGS catalyzed reaction is an asymmetric condensation of two molecules of the substrate 5-aminolevulinic acid (ALA). All organisms that carry out respiration, photosynthesis, or methanogenesis require PBGS, and the enzyme is remarkably conserved throughout evolution . The metabolic pathway from porphobilinogen to the tetrapyrroles is phylogenetically variable but universally populated by photoreactive intermediates whose accumulation can be toxic [3–4]. Thus, as part of the control of tetrapyrrole biosynthesis, PBGS evolved an allosteric regulation mechanism. Unexpectedly, PBGS allostery involves alternate, functionally distinct multimeric assemblies whose architectures are so different as to require multimer dissociation as part of the interconversion between active and inactive assemblies . Novel aspects of the allosteric regulation of PBGS led us to define a morpheein model of allostery (described elsewhere ); proteins that use this allosteric mechanism display properties that are characteristically different from proteins that follow the classic Monod-Wyman-Changeux and Koshland-Nemethy-Filmer models . Herein, we describe the phylogenetically variable allosteric regulation of PBGS; presumably these variations evolved due to the considerably different environments that organisms inhabit and the phylogenetic variation in the subcellular location of PBGS. In most species PBGS is located in the cytosol [8–9]. However, in plants it is in the chloroplast , and in apicomplexan parasites it is in the apicoplast .
PBGS is encoded by a single gene and each PBGS multimer is composed of multiple copies of the same protein. Each PBGS subunit consists of a ~300 residue αβ-barrel domain, which houses the enzyme active site in its center, and a >25 residue N-terminal arm domain (Fig. 1) . Allosteric regulation of PBGS can be described in terms of the orientation of the αβ-barrel domain with respect to the N-terminal arm domain . These two domains undergo a dramatic reorientation around a hinge region in the transition between active and inactive multimers (Fig. 1). Like most αβ-barrel enzymes, substrate access to the active site is mediated by a mobile loop that serves as an active site lid; the lid of each subunit comes from the same subunit as the rest of the active site. Allosteric regulation of PBGS can be described in terms of intersubunit interactions that assist in stabilizing an ordered conformation of the active site lid . These interactions involve the N-terminal arm domain from a subunit that is different from the subunit contributing the active site. In the active multimeric state, the N-terminal arm of one subunit interacts with the αβ-barrel domains of two other subunits. Similarly, each αβ-barrel interacts with the N-terminal arm of two adjacent subunits. One of these interactions helps to stabilize a “closed” conformation of the active site lid; this is the A/B interaction of Figure 2. The other interaction restricts solvent access from the other end of the αβ-barrel; this is the A/F interaction in Figure 2. In the inactive multimeric state, the N-terminal arm domain is not involved in the lid-stabilizing A/B subunit-subunit interaction. In the crystal structure of the inactive assembly, the active site lid is disordered (see Fig. 1 and Fig. 2B).
As described above, allosteric regulation of PBGS fits a familiar theme; the transition between active and inactive forms involves a conformational change that affects the traffic of ligands into and out of the enzyme active site. The initially unexpected aspect of PBGS allostery is that the active multimer is an octamer (8mer), the inactive multimer is a hexamer (6mer*), and the transition between 8mer and 6 mer* requires dissociation to a dimer. The dimer exists in an equilibrium of conformations, one of which can assemble to the 8mer, another of which can assemble to the 6mer*. The shorthand equation for this phenomenon is 8mer 2mer 2mer* 6mer*, where the oligomer-determining conformational change (2mer 2mer*) is a reorientation of the N-terminal arm domain of each subunit with respect to the αβ-barrel domain of the same subunit (seen most clearly in Fig. 1B). Steric considerations prevent this reorientation from occurring in either the 8mer or 6mer*, thus dissociation is an essential part of the allosteric mechanism.
Many PBGS proteins can participate in the quaternary structure dynamic equilibrium illustrated in Figure 3A, though the 2mer* and 6mer* assemblies are unavailable to some PBGS (Fig. 3B). The octameric assembly is supported by PBGS crystal structures from metazoa, fungi, bacteria, and alveolata (e.g. [11, 13–16]). The bacterial and alveolata structures all contain an allosteric magnesium ion located at the aforementioned lid-stabilizing arm-to-barrel A/B interface (Fig. 3B and and6B6B below) [2, 13–15]. An extensive phylogenetic analysis determined that the allosteric magnesium binding site is present in PBGS from nearly all species except metazoa and fungi . A multiple sequence alignment of the PBGS discussed in this review is provided in Figure 4 with key structural features highlighted. Before we appreciated the significance of the smaller alternate PBGS multimers, native PAGE was used to show that addition of magnesium to E. coli PBGS induces assembly to 8mer (Fig. 5A) . The 6mer* assembly is supported by the crystal structure of the human PBGS variant F12L and by analytical ultracentrifuge studies of both human and plant PBGS [5, 18]. In fact, native PAGE (Fig. 5B) and analytical ultracentrifugation have been used to show that removal of magnesium from plant PBGS promotes formation of the hexamer [5, 17]. The existence of dimeric assemblies is supported by size exclusion chromatography (SEC) and native PAGE for PBGS proteins from various species [19–20], but our understanding of the physiologically relevant dimer architecture has evolved in the past decade; furthermore the interconversion pathway may not be phylogenetically conserved.
Figure 3A illustrates two alternate pathways for interconversion of hexamer and octamer; the upper pathway shows the transition through “hugging” and “detached” dimers that are the asymmetric units of human PBGS crystal structures, while the lower pathway shows the transition through the alternate “pro-octamer” and “pro-hexamer” dimers. The identity of the dimeric species, which comprise less than 1% of the total human PBGS in solution , remains an unknown but intriguing question. Thermodynamic rationale for favoring one pathway over the other is discussed extensively in , and summarized below.
The hugging and detached dimers were originally considered as the solution structures for the PBGS dimers and were depicted as such in many of our earlier publications (e.g. [5–7, 18, 21–24]. Dissociation of an 8mer or 6mer* to hugging or detached dimers (respectively) involves breaking the subunit-subunit interface wherein the N-terminal arm of one subunit excludes solvent access from the back of the αβ-barrel of another subunit (A/F interface of Fig. 2A). Breaking this arm-to-back-of-barrel interface exposes a hydrophobic patch dominated by Trp19. In support of the importance of Trp19 to multimerization, the human PBGS variant W19A is dimeric and cannot assemble to either 8mer or 6mer* . We note that the residues that participate in this arm-to-back-of-barrel (A/F) interaction are not phylogenetically conserved. For instance, in PBGS sequence alignments (e.g. Fig. 4), Trp19 of human PBGS appears as an insertion in the N-terminal arm domain, which itself has a much lower sequence conservation than the αβ-barrel domain . The primary conformational change between the hugging and detached dimers is observed as a rotation that changes the orientation of the N-terminal arm (Fig 3A). In the “arm down” conformation of the hugging dimer, the hugging interaction of the arm with the αβ-barrel of its partner monomer is evident. In the context of the 8mer, additional interactions that may stabilize the secondary structure of the arm also derive from a third subunit (subunit F in Figs. 2A and and3A).3A). The “arm up” conformation of the detached dimer places the arm distal from the αβ-barrel of its partner monomer, thereby leaving this arm with no protein-protein stabilizing interactions. This lack of stabilizing interactions for the N-terminal arm conformation in the detached dimer, coupled with exposed hydrophobic patches in both hugging and detached dimers lead us to hypothesize that oligomeric interconversion proceeds through the pro-octamer and pro-hexamer dimers as discussed below.
Dissociation via the pro-octamer and pro-hexamer dimers exposes the large A/B subunit-subunit interface, which has two components. One component of the A/B interface is a symmetric barrel-barrel interaction that is highly conserved, dominated by hydrophilic residues and charge-charge interactions, and for which water-containing crystal structures from different species all contain ordered water molecules whose locations are phylogenetically conserved. Components of this interface include reciprocal bonds between the universally conserved ion pair Asp259 and Arg309, and the hydrogen bonded pair Tyr257 and Glu302 (numbering corresponds to human PBGS). For human PBGS the barrel-barrel interface does not contain any aromatic-aromatic intersubunit interactions, nor does it contain a hydrophobic patch like that described above for the arm-to-back-of-barrel interface. The second component of the A/B interface is the hugging arm-to-barrel interaction that stabilizes the ordered conformation of the active site lid. Although phylogenetically variable, this arm-to-barrel interaction is also hydrophilic, significantly hydrated in structures containing modeled waters, and includes the binding site for the aforementioned allosteric magnesium (see Fig. 3B). Metazoan and fungal PBGS, which do not have the magnesium binding site, contain instead a spatially equivalent arginine guanidinium group (Fig. 6). Removal of this arginine (e.g. human PBGS variants R240A and R240W [22–23]) results in a protein that favors assembly to 6mer* relative to wild type, which favors assembly to 8mer. We have argued that dissociation along the A/B interface is energetically feasible because the dissociating surfaces are hydrophilic and already hydrated in 8mer and 6mer* . The same changes in phi/psi angles that produce the arm-up and arm-down conformations of the hugging and detached dimers are reflected in the pro-octamer and prohexamer dimers as a twist at a hinge between the N-terminal arm and the αβ-barrel domains. This twist changes the orientation of the two αβ-barrels relative to each other (best seen in Fig. 3A) and, importantly, maintains the hydrophobic arm-stabilizing arm-to-back-of-barrel interactions of the A/F interface for both the pro-octamer and pro-hexamer dimers. Further support of the physiologic relevance of the pro-octamer dimer comes from the X-ray crystal structure of PBGS from Toxoplasma gondii, as shown in Fig. 3B and discussed below.
As noted above, the N-terminal arm of PBGS, whose position is of pivotal importance to multimer architecture, has significant phylogenetic variability. When first characterizing the oligomeric equilibrium, we found the 8mer 2mer 2mer* 6mer* pathway to be common to PBGS from both humans and plants, even though the N-terminal sequences vary (see Fig. 4). Thus, when we undertook characterization of PBGS from bacterial and eukaryotic pathogens, we were surprised to discover that PBGS from many of these species does not sample the 2mer* 6mer* components of the equilibrium. Two of these species are the human pathogens Vibrio cholerae and Toxoplasma gondii, where our work with recombinant PBGS provided no evidence for the hexamer as a component of the equilibrium of quaternary structure isoforms . Native PAGE of PBGS from these species shows predominantly octamer, with low levels of dimer present. Sequence alignment shows that these two species of PBGS are among a relatively small number that also includes a short sequence C-terminal to the common αβ-barrel domain . The recently solved crystal structure for T. gondii PBGS revealed that a 13 amino acid C-terminal extension (see Fig. 4) forms a domain-swapped intersubunit β-sheet (Fig. 3B) that wraps around the hinge region of the pro-octamer dimer and prevents rotation to the pro-hexamer dimer, thus preventing hexamer formation (Fig. 3B) . We posit that for other species where PBGS has a C-terminal extension, a similar intersubunit β-sheet prevents population of the hexamer. In PBGS where the pro-octamer dimer is tethered by a β-sheet, it appears that this is the architecture of the dimeric component of the quaternary structure equilibrium. For these species, it may have been evolutionarily advantageous to keep PBGS in an active octameric conformation. Alternatively, allosteric regulation may be achieved through a simple equilibrium between 8-mer and pro-octamer dimer. Although the C-terminal extension provides a rationale for why some species of PBGS do not populate the 2mer* 6mer* portion of the quaternary structure equilibrium, it does not explain why some other PBGS without this extension also fail to form the hexamer. One well characterized example is PBGS from Pseudomonas aeruginosa, where we have found small molecules that stabilize a dimeric assembly (see below) .
Inborn errors of metabolism often arise from defects in protein structure and function. ALAD-porphyria is a very rare inborn error of metabolism that arises from mutations to the gene encoding PBGS [26–27]. Wild type levels of PBGS activity are far in excess of that required for heme biosynthesis . Consequently, individuals with clinical ALAD porphyria result from compound heterozygotes who inherited one defective gene from each of two asymptomatic parents. With insufficient PBGS activity, these patients can experience elevated levels of ALA, a structural analog of the neurotransmitter gamma-amino butyric acid, resulting in adverse neurologic consequences. There are eight ALAD-porphyria associated variants that have been documented, one of which is the aforementioned F12L. When heterologously expressed, F12L appears as an obligate hexamer with a pH rate profile that is dramatically different from the wild type protein (Fig. 7A). However coexpression of the wild type and F12L variant leads to production of both 8mer and 6mer* hetero-multimers; the pH rate profile of the hetero-6mer* is included in Figure 7A and highlights its metastable character (see below). The positions of the seven additional ALAD-porphyria associated variants are widely distributed throughout the protein and, like F12L, their positions would not be predicted to affect the quaternary structure equilibrium (Fig. 7B). However, when heterologously expressed in E. coli, each of the ALAD-porphyria associated variants exhibited an increased mole fraction of hexamer relative to the wild type protein . This disease association was the first indication of the physiologic relevance of the human PBGS quaternary structure equilibrium. Later studies revealed drug-derived allosteric inhibitors of human PBGS (see below) [28–29].
As a nearly universally essential enzyme with a highly conserved active site, PBGS is not a canonical target for the development of antimicrobials and/or herbicides. However, allosteric sites can be much more phylogenetically variable than active sites and present drug development opportunities. Phylogenetic variations in the PBGS quaternary structure equilibrium suggests that we can target allosteric regulation of PBGS in a species-specific manner for development of antimicrobials and herbicides. The classic definition of an enzyme's allosteric regulator is an entity that binds at a site other than the active site and alters the rate of the catalytic reaction. The earliest revealed allosteric regulators were metabolites, which can be considered intrinsic. Alternatively, extrinsic factors are exogenous entities such as drugs or toxins that bind outside the enzyme active site but change the protein structure in such a way as to inhibit activity. Phylogenetic variation in PBGS allostery leads us to frame discussion of PBGS allosteric regulators in terms of intrinsic and extrinsic factors.
One intrinsic allosteric regulator is the aforementioned allosteric magnesium ion, which is generally present in PBGS from species other than metazoa and/or fungi. The allosteric magnesium ion lies at the highly hydrated (A/B) interface of two prooctamer dimers (Fig. 6A). It has only one amino acid side chain-derived first coordination sphere ligand (always an aspartate; Asp231 in E. coli PBGS), with the other five ligands being water molecules. Second coordination sphere ligands include the guanidinium group of an arginine that comes from a different subunit (Arg11 of E. coli PBGS), which is in the N-terminal arm of an adjacent pro-octamer dimer. Thus, this magnesium appears to be easily dissociable and we have shown by native PAGE and analytical ultracentrifugation that hexamers accumulate when magnesium is removed in vitro (see Fig. 5B) [5, 18]. In plants, where PBGS is located in the chloroplast, the Kactivation for the allosteric magnesium is ~ 2.5 mM . We posit that magnesium activation of PBGS in plants is physiologically relevant because one of the earliest events when developing chloroplasts are exposed to light is a change in the magnesium concentration from <1 mM to >10 mM . Accumulation of photoreactive chlorophyll precursors in the dark can be deadly; for instance, application of the PBGS substrate ALA to plants in the evening has been used as a herbicide [32–36]. The physiologic relevance of the PBGS allosteric magnesium in other species is unclear, particularly in light of the fact that magnesium in the millimolar range is required for many biochemical structures and metabolic processes (e.g. oligonucleotide structure, phosphoryl transfer reactions, ligases).
It is not common to consider protons (or hydronium ions) as allosteric regulators. However, in the case of PBGS, side chain protonation at locations other than the active site have been shown to affect the quaternary structure equilibrium, and thus to affect the rate of the PBGS catalyzed reaction. Here we discuss the arginine residue, Arg240 of human PBGS, whose guanidinium group is spatially and functionally equivalent to the allosteric magnesium (Fig. 6B). This arginine is conserved in PBGS from metazoa and fungi. The importance of Arg240 to the stability of the human PBGS 8mer is illustrated by the behavior of our designed variant R240A and the naturally occurring ALAD-porphyria associated variant R240W [22–23]. Although wild type human PBGS purifies at an 8mer:6mer* ratio of 95:5, this ratio for R240W is 20:80, and for R240A is 10:90. We posit that this shift in quaternary structure equilibrium is related to the role of Arg240 in stabilizing the interaction between two pro-octamer dimers; the guanidinium group of Arg240 forms a hydrogen bond with the carbonyl oxygen of Ser5 of a neighboring pro-octamer dimer. In the obligate 6mer* F12L variant, the displaced N-terminal arm (Fig. 6C) obviously also forbids the intersubunit hydrogen bond. Of course, this single hydrogen bond is not the only determinant for 8mer stability. Rather it is one of a multitude of factors that must all be present to maintain human PBGS as the 8mer as described in the following paragraphs.
The experimentally determined behavior of R240A teaches us about the fragile nature of the human PBGS 8mer, maintenance of which requires a multitude of factors illustrated in Figure 8A. Each of these factors contributes to stabilizing the ordered or “closed” conformation of the active site lid. Figure 8B illustrates an experiment wherein the isolated R240A 6mer* is dialyzed for 24 h against assay buffer containing the substrate ALA, at both pH 7 and pH 9, and samples are removed at various times to determine the 8mer:6mer* ratio by native PAGE . Regardless of the pH, dialysis against substrate draws the equilibrium from 6mer* toward 8mer, though the extent of conversion is pH dependent. Stabilization of the active site lid, which occurs when the carboxyl group of A-side ALA interacts with two lid-derived arginine residues (see Fig. 8A), favors 8mer formation. PBGS crystal structures lacking a carboxyl-containing A-side ligand show a disordered active site lid. Raising the pH of the dialysis buffer from pH 7 to pH 9 causes a partial deprotonation of 8mer-stabilizing side chains, which may include the lid arginine residues, as well as Arg240, and reduces the ALA-induced conversion to 8mer. This pH-dependent decrease in the 8mer:6mer* ratio is also reflected in the basic arm of the human PBGS pH rate profile (see Fig. 7A) .
In the second phase of the experiment shown in Figure 8B, the dialysis cassette is transferred to the same buffer in the absence of substrate. Native PAGE demonstrates a time-dependent shift of the equilibrium back toward 6mer*. This slow transition back to 6mer* as the substrate induced active site lid stabilization dissipates illustrates the metastable nature of the human PBGS multimers. Another factor that is essential to binding A-side ALA to human PBGS is the active site zinc ion ; exposing the protein to the zinc chelator 1,10-phenanthroline also causes accumulation of the 6mer* (unpublished data). As illustrated in Figure 8A, there is one additional intersubunit interaction that appears essential to maintain the integrity of the 8mer. This is between the side chain of Cys223 and the backbone oxygen of Phe12 on the neighboring pro-octamer dimer. Although we do not yet fully understand why, we believe that the F12L mutation compromises this interaction and thus results in observation of 6mer* as the only higher order multimer.
The observations detailed above depict the human PBGS 8mer as relatively fragile, requiring many different interactions to maintain 8mer structural integrity. We posit that the half-of-the-sites reactivity seen in mammalian PBGS is a consequence of this frailty. Data supporting half-of-the-sites in mammalian PBGS stems from active site ligand trapping studies (e.g. [37–39]), and the stoichiometry of active site metal ion requirement (e.g. [40–42]). In the event that human PBGS were without A-side substrate (or product) bound at the enzyme active site, active 8mer stability is compromised. Therefore, the protein has evolved a half-of-the-sites mechanism where product is not released from one hemisphere of 8mer until the first substrate (P-side ALA) binds to the other hemisphere of 8mer. This ultimate form of negative cooperativity allows the protein to remain in the 8mer state. It is intriguing to hypothesize that maintenance of quaternary structure integrity may be a common theme for proteins that exhibit half-of-the-sites reactivity. This phenomenon could be considered a form of allostery, where binding events at one active site affect the reactivity of a neighboring active site.
Inspection of the PBGS 6mer* reveals a surface cavity that is not present in the 8mer (Fig. 9A). Small molecule binding to this phylogenetically variable cavity was proposed to stabilize 6mer* of the targeted PBGS and consequently inhibit activity. We have called such allosteric regulators morphlocks because they lock PBGS in a specific morpheein form (6mer*). Although we have yet to find intrinsic ligands that act as 6mer*-stabilizing morphlocks, both computational docking and in vitro library screening have revealed a significant number of morphlocks as described below.
A computational docking approach was used to find morphlocks for PBGS from plants (Pisum sativum, a.k.a. pea), human, and the human pathogen Yersinia enterocolitica. The docking was directed to the 6mer*-specific surface cavity (Fig. 9A and 9B) and the small molecule library was a collection of ~300,000 drug like compounds that were reportedly commercially available from Life Chemicals Inc. In the case of pea PBGS and Y. enterocolitica PBGS, the docking targets were carefully constructed protein structure models, and in the case of human PBGS it was the F12L crystal structure [24, 28, 43]. Docking was used to refine the large library down to a number that we could afford to purchase and test in vitro. In each case 100 compounds were selected from the top scoring 1% of the docked molecules. In this case, docking to a crystal structure yielded a larger number of morphlocks than docking to a protein structure model [24, 28]. One such inhibitor is morphlock-1 (2-oxo-1,2-dihydro-benzo(cd)indole-6-sulfonic acid(2-hydroxy-2-[4-nitro-phenyl]-ethyl)-amide), which like most of our 6mer*-stabilizing PBGS inhibitors shows the predicted species specificity, inhibiting its targeted pea PBGS with an IC50 of ~1 μM while no inhibition is seen for human PBGS at a morphlock-1 concentration of 100 μM. Figure 9C illustrates an activity stained native PAGE of pea PBGS which establishes that morphlock-1 binds to the hexamer, inhibits enzyme activity, and is bound sufficiently tightly to prevent the substrate ALA drawing the equilibrium back to the octamer.
The first set of 6mer*-stabilizing human PBGS morphlocks arose from the computational docking study described above (and depicted in Fig. 9 for pea PBGS). Surprisingly, one of the identified human PBGS morphlocks (5-chloro-7-(diethylaminomethyl)quinolin-8-ol) is a chemical homolog to a drug currently in clinical use (5-chloro-8-hydroxy-7-iodoquinoline, a.k.a. clioquinol); purchase and testing of the drug confirmed that it is also a 6mer*-stabilizing human PBGS morphlock. The literature suggests that side effects of this drug mimic a porphyric attack  and we established that 6mer*-stabilizing inhibition of this drug was more potent with ALAD-porphyria associated variants, which are inherently more prone to form the 6mer . This result suggested significant physiologic relevance to the allosteric regulation of the human PBGS quaternary structure equilibrium by extrinsic factors, such as drugs and/or environmental contaminants. To address this hypothesis, a medium throughput native PAGE mobility shift assay was used to screen the Johns Hopkins Clinical Compound Library and an alarming 1% of the components in that library were found to act as 6mer*-stabilizing inhibitors of human PBGS in vitro . One notable example is the widely available nonsteroidal anti-inflamatory diclofenac (a.k.a. Voltaren). Porphyrias in general are highly episodic diseases and the contributing factors are not well understood. One contributing factor is exposure to lead, which inhibits human PBGS by replacing the active site zinc. It is possible that exposure to small molecule 6mer*-stabilizing human PBGS allosteric inhibitors, such as the drugs we have identified, could be another contributing factor.
Despite our remarkable success in the identification of 6mer*-stabilizing PBGS inhibitors, this approach to PBGS inhibitor discovery is not applicable to all species. Our studies of PBGS from the human pathogens Pseudomonas aeruginosa, Toxoplasma gondii, and Vibrio cholerae indicate that these proteins do not sample the 2mer* and 6mer* components of the equilibrium. Thus, discovery of allosteric regulators of these species of PBGS is directed at small molecule inhibition of 8mer assembly. Again a docking approach was taken targeting a site predicted to prevent 2mers from assembling to 8mers, and some success has been obtained. Unsure of the structure of the physiologically relevant dimer of P. aeruginosa PBGS, docking was carried out against the interface between two hugging dimers as well as the interface between two pro-octamer dimers. Small molecules that prevent dimer assembly to 8mer (as determined by native PAGE) were discovered only in the latter case . In this case, however, addition of ALA was sufficient to draw the equilibrium back toward the active 8mer. Thus, none of these oligomerization disabling compounds were found to inhibit the bacterial PBGS catalytic activity. Efforts toward small molecule modulation of the quaternary structure equilibrium of T. gondii and V. cholerae PBGS are ongoing.
The morpheein model of allostery exemplified by PBGS adds an additional layer of understanding to potential mechanisms for regulation of protein function and complements the increased focus that the protein science community is placing on protein structure dynamics. The physiologic importance of phenomena such as alternate protein conformations, alternate oligomeric states, and transient protein-protein interactions are well-established in cell signaling (i.e. oligomerizing cell surface receptors), virology (i.e. the existence of multiple oligomers of identical virus coat proteins), and amyloid disease. The morpheein model of allostery illustrates how the dynamics of these phenomena can be harnessed for allosteric regulation of catalytic activity. The well-characterized PBGS example illustrates how a powerful dissociative allosteric mechanism has been adapted throughout evolution to provide phylogenetically variable control of an essential pathway. Our work on PBGS from various species illustrates how activity can be regulated via its oligomeric equilibria in response to a changing intracellular environment (intrinsic modifiers). Our in silico and in vitro screening has identified inhibitors that function by shifting the PBGS oligomeric equilibrium, thus highlighting the sensitivity of this allosteric mechanism to small molecules not native to the cell (extrinsic modifiers).
The quaternary structure equilibrium of PBGS provides an opportunity to address new aspects of protein folding and assembly. The dissociative nature of the 8mer 2mer 2mer* 6mer* equilibrium raises intriguing questions about the thermodynamics of multimer dissociation. For instance, we have not found rigorously benchmarked resources for determining the thermodynamic value of hydrating newly dissociated protein interfaces that take into account waters that are present prior to dissociation. Furthermore, only some protein crystal structures contain modeled water that can contribute to this calculation. These uncertainties confound theoretical identification of which interfaces are maintained or sacrificed when a higher-order oligomer dissociates. Such structural questions gain importance when searching for allosteric inhibitors designed to interfere with assembly of an inactive PBGS 2mer to the active 8mer. However, we intend to pursue experimental approaches that can help clarify the PBGS dissociative pathway.
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