Biomedical subjects

Linda Stith

Publications and source records attributed to Linda Stith.

4 recordsLinked to original sources

PubMed2005

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins).

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.

Amino Acid Substitution↗

PubMed2005

Substrate-induced interconversion of protein quaternary structure isoforms.

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. 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.

Humans↗

PubMed2003

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

Allosteric Site↗

PubMed2003

A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase.

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.

Animals↗