Dissociating allosteric enzymes are well established (Traut, 1994
) and provide opportunities for small molecule inhibition by interference with the assembly process. If one couples dissociating enzymes with conformational flexibility (James and Tawfik, 2003
), one can achieve a system wherein subunits of an oligomer of one symmetry may dissociate and then change conformation such that they can only assemble to an oligomer of different symmetry. In this case, it may be possible for small molecules to trap one oligomeric assembly and prevent equilibration to the alternate assembly. The morpheein model for allosteric regulation of protein function describes such a situation wherein proteins can exist as an ensemble of physiologically significant and functionally distinct alternate quaternary assemblies () (Jaffe, 2005
). The current paper addresses the hypothesis that small molecules can act as inhibitors of such proteins by selectively binding to and stabilizing the less active assembly ().
Proteins that can exist as an equilibrium of alternate quaternary structure assemblies (morpheeins) provide a structural foundation for allosteric regulation of protein function
The morpheein model for allostery is distinct from the classic Monod-Wyman-Changeau and Koshland-Nemanthy-Filmer models for allostery (Koshland et al., 1966
; Monod et al., 1965
), both of which contain the assumption of a conserved oligomeric assembly throughout the allosteric transition. This implies a fixed stoichiometry, which need not hold true for homo-oligomeric proteins that function as morpheeins. The distinguishing feature between the morpheein model for allosteric regulation and both classic models is that the former must
involve a dissociation event and a conformational change in the dissociated state; it may also involve reassembly into a functionally distinct alternate oligomer. The structurally distinct quaternary assemblies available to a morpheein present a previously unforeseen opportunity for allosteric chemical inhibition. The mode of action for the proposed quaternary structure-trapping agent is to bind to an oligomer-specific surface cavity and draw the equilibrium toward the targeted oligomeric form (). Like many allosteric sites, the novel small molecule binding sites are likely to be more phylogenetically variable than enzyme active sites, allowing one to target universally essential enzymes or proteins for drug discovery.
The prototype morpheein, porphobilinogen synthase (PBGS, EC 22.214.171.124, a.k.a. 5-aminolevulinate dehydratase), catalyzes a fundamental step in the biosynthesis of tetrapyrrole pigments, an activity that is essential to all organisms that carry out respiration, photosynthesis, or methanogensis. PBGS has been shown to exist in an equilibrium of high activity octamers and low activity hexamers whose interconversion is at the level of two different dimer conformations (Breinig et al., 2003
; Selwood et al., 2008
; Tang et al., 2006
; Tang et al., 2005
) (). Ligand binding to the active site draws the equilibrium toward the octamer. A consequence of this equilibrium of oliogmeric assemblies is a protein concentration dependent specific activity (Kervinen et al., 2000
), which we now interpret to reflect a low-activity hexamer dissociating to dimers, changing configuration, and then re-associating to an active octamer (or vice versa
). Crystal structures reveal the alternate assemblies of PBGS () (Breinig et al., 2003
; Frankenberg et al., 1999
) and show that each monomer is comprised of an αβ-barrel domain and an extended N-terminal arm with phylogenetically variable length; the orientation of the arm with respect to the barrel is a determinant of the quaternary structure assembly. The crystallographic asymmetric units are the illustrated dimers. For both the octamer and the hexamer the active site is located in the center of the αβ-barrel. The N-terminal arm differentially participates in subunit-subunit interactions in the octameric and hexameric assemblies. It is the difference in intersubunit arm-to-barrel interactions that contributes to dramatic kinetic differenced between the octameric and hexameric assemblies (Jaffe, 2004
). Phylogenetic variations in the N-terminal arm sequence result in a phylogenetic variation in the thermodynamics and kinetics of the equilibration of PBGS oligomers.
Porphobilinogen synthase (PBGS) is the prototype morpheein ensemble
The physiologic relevance of the octamer-hexamer equilibrium illustrated in is established for PBGS from both humans and plants. In humans, the disease ALAD porphyria arises from mutations to the gene encoding PBGS (Gross et al., 1998
). There are eight known human mutations associated with ALAD porphyria; all eight are associated with an increased propensity of the protein to exist as the inactive hexameric assembly (Jaffe and Stith, 2007
). In plants, PBGS resides in the chloroplast (Boese et al., 1991
) and is established to contain an allosteric magnesium binding site (Kervinen et al., 2000
). Magnesium binding to this site facilitates the hexamer to octamer transition by stabilizing a subunit interface that is present in the octamer but not in the hexamer () (Breinig et al., 2003
). During the greening process in plants the resting magnesium concentration in the chloroplast is below 1 mM and the resultant low PBGS activity helps prevent the accumulation of phototoxic chlorophyll precursors. Upon exposure to light the chloroplast magnesium concentration increases to ~10 mM (Walker, 1976
); appropriately, the Kd
for the allosteric magnesium of the green plant Pisum sativum
(pea) PBGS is 2.5 mM (Kervinen et al., 2000
). The physiologic relevance of the quaternary structure equilibrium of PBGS from human pathogens has not yet been established. However, preliminary results on PBGS from several pathogens show the protein concentration dependent specific activity indicative of an equilibrium of quaternary structure assemblies under native conditions (unpublished results).
The current study was undertaken to establish whether species-selective inhibition of PBGS could be accomplished by perturbing its quaternary structure equilibrium. The PBGS from pea was selected as the inhibitor target. This well-characterized protein exhibits protein concentration dependent specific activity and is available in large quantities (Kervinen et al., 2000
). A cavity on the surface of the hexameric assembly (inactive oligomer) serves as a putative small molecule binding site (); this site is not present in the octamer (active oligomer). A quaternary-structure-perturbing inhibitor for pea PBGS would function by binding to the illustrated site in the hexamer (), thereby drawing the equilibrium toward the stabilized inactive form and reducing the total activity. The sequence of PBGS from both plants and human pathogens differ from the human protein at this proposed small molecule binding site (). Due to these phylogenetic sequence differences, an inhibitor selected for this site in plant or pathogen PBGS would not be expected to affect the activity of human PBGS. This is in stark contrast to an active site directed PBGS inhibitor as the active site residues are highly conserved (Jaffe, 2003
docking using the GLIDE program (Halgren et al., 2004
) was utilized to identify a suite of small molecules predicted to bind to the hexamer-specific binding site of a homology model of pea PBGS. A selection of these molecules were purchased and tested in vitro
for their ability to stabilize the hexameric form of pea PBGS and to inhibit enzyme activity. One potent inhibitor, which drove the pea PBGS oligomeric distribution dramatically toward the hexamer, was identified from the screen. This compound, given the name morphlock-1, did not inhibit the activity of human PBGS, nor did it alter the quaternary structure equilibrium of PBGS from humans, Drosophila melanogaster
, Pseudomonas aeruginosa
, or Vibrio cholerae
. Thus, we have demonstrated that perturbation of a morpheein equilibrium of non-additive quaternary structure assemblies (see ) is a viable approach for the development of species-specific drugs with novel modes of action.