Single Amino Acid Mutations Alter the Distribution of Human Porphobilinogen Synthase Quaternary Structure Isoforms (Morpheeins)

Abstract

Evidence is building to support the notion that the porphobilinogen synthase (PBGS 2 ; EC 4.2.1.24) family of enzymes can exist as an equilibrium of quaternary structure isoforms, denoted morpheeins (1-3). Morpheeins comprise an equilibrium ensemble of protein structures wherein a protein monomer can exist in more than one conformation, and each monomer conformation dictates a functionally different quaternary structure of finite multiplicity. Morpheeins have been proposed to provide a structural foundation for allosteric regulation, cooperativity, and hysteresis in some proteins (2). As such, the energetic difference between morpheeins of a given protein must be small. The propensity of PBGS to assume various morpheein structures and the rates of PBGS morpheein interconversion are highly species-dependent. The stable morpheeins of human PBGS are the octamer, found for the wild-type protein, and the hexamer, first seen for the naturally occurring mutation F12L (1). Coexpression of human wild-type PBGS and F12L generates a population of PBGS proteins composed of hetero-octamers and heterohexamers, each of which contains a mixture of Phe 12 -and Leu 12 -containing chains (1). The structure and composition of these hetero-oligomers are stable during storage, but the molecular motions resulting from catalysis favor formation of the octamer with an accompanying disproportionation of Phe 12 -containing chains into the octamer (3).The remaining hexamer has an increased proportion of Leu 12 -containing chains (3). The physical basis for the thermodynamic propensity of Leu 12 -containing chains to form the hexamer remains unclear, but examination of the structure of human PBGS suggests that other single amino acid mutations might affect the folding and assembly of the protein to favor structures other than the octamer. In this study, we report on alterations of two amino acids (Arg 240 and Trp 19 ) that were chosen based on an analysis of the structures and subunit interactions seen in human octameric and hexameric PBGS, for which the assemblies are shown in Fig. 1. Each human PBGS subunit is composed of a 306-amino acid TIM-like ␣␤-barrel and a 24-amino acid N-terminal arm. The conformational difference between the monomer that assembles into the octamer and the monomer that assembles into the hexamer is a dramatic reorientation of the arm with respect to the barrel; for the F12L hexamer, this reorientation occurs at Thr 23 . In both the octameric and hexameric assemblies, two monomers come together to form a dimer with a conserved barrel-barrel interface. The dimer that assembles into the octamer is called a hugging dimer (Fig. 1a), whereas the dimer that assembles into the hexamer is called a detached dimer (Fig. 1b) (1). The difference between these two dimers is the presence or absence of a "hugging" interaction between the N-terminal arm of one subunit and the ␣␤-barrel of the adjacent subunit of the dimer. The "arm-hugging-barrel" interaction of PBGSs from plants, Archaea, and most Bacteria is stabilized by an all...

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

“…2). Experimentally eradicating this interaction in human PBGS by the mutation W19A resulted in a dimeric protein that could make neither octamer nor hexamer (24).…”

“…3) (34). Those PBGS that do not have the allosteric magnesium site have an arginine residue whose guanidinium group is spatially equivalent to the magnesium ion, and which has been shown to stabilize the octamer (24). TgPBGS does not have the cysteine-rich site but has the allosteric magnesium binding site (as defined by comparative sequence information); this is characteristic of PBGS from eukaryotes in the phyla Alveolata, Rhodophyta, Crytophyta, Glaucocystophyceae, Stramenopiles, and Verdiplantae and a subset of bacteria (12).…”

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

“…Full-length rat PAH was expressed in BLR-DE3 cells using a 2-d expression as described (31), with the exception that ferrous ammonium sulfate (0.2 mM) was added for the expression phase of the procedure. Protein was purified as previously described (12).…”

Allosteric Inhibition of Human Porphobilinogen Synthase

“…Expression and Purification of PBGS-PBGS from human (N59/C162A wild type variant, E89K, and A274T) were expressed and purified as previously reported (3,10).…”

“…No significant change in PBGS oligomer distribution was observed for any of the compounds when using a 2-h incubation at pH 7. Although the high activity octameric assembly dominates the wild type human PBGS quaternary structure equilibrium at neutral pH, at higher pH values the hexamer is more highly favored (1,10). Thus, the gel shift screen was repeated at pH 8 where the intrinsic population of the wild type hexamer is measurable, and the probability of trapping this species is, thus, increased.…”