Substrate-induced Interconversion of Protein Quaternary Structure Isoforms

Abstract

(2003) Nat. Struct. Biol. 10, 757-763). The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.A recent description of a rare human allele of porphobilinogen synthase (PBGS) 1 revealed that the enzyme can exist as two alternate quaternary structures wherein the oligomeric state is dictated by distinctly different structures for their component crystallographic asymmetric unit (1). The monomer is an ␣␤-barrel protein with a 23-residue N-terminal arm. The octameric form is comprised of four "hugging dimers" (Fig. 1a), whereas the hexameric form is comprised of three "detached dimers" (Fig. 1b). In both oligomeric assemblies there are invariant barrel-to-barrel subunit interactions within the dimers and quasiequivalent arm-to-bottom-of-barrel subunit interactions between adjacent dimers (Fig. 1). However, the octamer has a unique "hugging" arm-to-barrel interaction that is not seen in the hexamer.Preparations of heterologously expressed wild type human PBGS predominantly contain octamer, but there is a small propensity to adopt the hexameric structure (1). In contrast, heterologous expression of the rare human variant, F12L, yields protein that purifies as the hexamer and does not readily take on the octameric structure. The homo-octameric wild type and homohexameric F12L forms of human PBGS exhibit dramatic differences in pH activity profile and in the kinetic K m values (Table I) (1) despite the fact that amino acid residue 12 does not interact directly with active site residues in either quaternary structure isoform (Fig. 1). However, the arm-tobarrel interface found in the hugging dimer provides substantial stabilization for the solvent-accessible face of the ␣␤-barrel wherein lies the active site. This region of the protein, which is disordered in the hexamer, includes an ϳ11-amino acid stretch that normally serves to gate the active site from solvent, also called the active site lid. With the active site more solventaccessible in the hexamer, a high external pH is required to drive the Schiff base formation that is necessary for catalysis (2); this contributes to the high pH required for F12L activity. The decreased affinity of F12L for substrate is attributed to the loss of interactions between the destabilized active site lid and the carboxyl moiety of the outermost substrate (3).Coexpression of wild type and F12L (WTϩF12L...

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii

“…S2B) Fig. 2A), comparable with previous reports for PBGS from other species (7,17,26,27). TgPBGS activity is also dependent on protein concentration, reaching maximal activity at ϳ10 g/ml (ϳ0.25 M) and an apparent K d of ϳ0.04 M (Fig.…”

“…Comparison with standard marker proteins indicates a molecular mass of ϳ323-kDa TgPBGS, consistent with an octameric structure (Fig. 3A), as observed in other PBGS enzymes (7,31). Unlike HsPBGS or PsPBGS, however, hexamers were not evident in TgPBGS, even as a shoulder of the main FIGURE 3.…”

“…PBGS enzymes are highly conserved metalloproteins, with well documented enzymatic and structural properties (3,4). Unusual characteristics include extensive variation in metal ion usage at the active and allosteric sites (5) and an unusually dynamic quaternary structure (6,7). Cytosolic (animal/fungal) PBGS enzymes are Zn 2ϩ -dependent, whereas the plastidic (plant/algal) PBGSs respond to Mg 2ϩ .…”

“…Sequence conservation and activity assays suggest that the latter contain two distinct Mg 2ϩ binding sites: one presumed to be analogous to the catalytic Zn 2ϩ in animal/fungal PBGS and an allosteric site present at an interdimer interface (5). Both the human (HsPBGS) (7) and Pisum sativum (PsPBGS) (6) enzymes exhibit allosteric regulation based on quaternary structure; homooctamers are highly active, whereas homohexamers are inactive; dissociation and conformational change at the dimer level is required for hexamer-octamer interconversion. Misregulation of the human PBGS quaternary structure equilibrium is associated with porphyric disease (8).…”

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

“…"Hugging" and "detached" dimers (pathway A) are observed as the asymmetric crystallographic units for wild type and mutant forms of human PBGS (PDB codes 1E51 and 1PV8, respectively (2,6)). The structure of the physiologically relevant dimer, and the pathway of hexamer-octamer conversion, remains unclear, however, as biochemical and biophysical tools demonstrate that interconversion between octameric and hexameric oligomers involves a conformationally flexible dimer (4,7). Analysis of wild-type human PBGS establishes that the hexamer is a pH-dependent component of the quaternary structure equilibrium and suggests that oligomer dissociation proceeds via the pro-octamer and pro-hexamer dimers (Fig.…”