Abstract
Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.
Highlights
Wherein a protein monomer can exist in more than one conformation, and each monomer conformation dictates a functionally different quaternary structure of finite multiplicity
Coexpression of human wild-type Porphobilinogen synthase (PBGS) and F12L generates a population of PBGS proteins composed of hetero-octamers and heterohexamers, each of which contains a mixture of Phe12- and Leu12-containing chains [1]
The “arm-hugging-barrel” interaction of PBGSs from plants, Archaea, and most Bacteria is stabilized by an allosteric magnesium (Fig. 2a), which has been seen in the crystal structures of PBGSs from Pseudomonas aeruginosa [4] and Escherichia coli [5] and which has been shown to facilitate the formation of the octamer [1, 6]
Summary
We posit that Arg240 provides stabilization for the human PBGS octamer via hydrogen-bonding interactions with Ser of the adjacent chain and that the human PBGS mutant R240A would fold and assemble into a hexamer. This hypothesis is proven to be correct. A second hypothesis relates to a dimer-dimer interface that is common to both the hexameric and octameric assemblies of human PBGS This interaction snuggles the N-terminal arm of the upper subunit of the first dimer against the bottom face of the ␣-barrel of the lower subunit of the second dimer (Fig. 3). The sum of the data on the human PBGS mutant F12L, which occurs naturally, and the synthetic mutants R240A and W19A demonstrate that there are single amino acid mutations in different parts of human PBGS that can dramatically destabilize the folding and assembly away from the native octameric state, yet some mutations allow interconversion between morpheein forms
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