Human porphobilinogen synthase (PBGS;E.C. 4.2.1.24) catalyzes the ancient and essential formation of the fundamental biological monopyrrole, porphobilinogen in the heme biosynthetic pathway. It is a zinc metalloenzyme and the target enzyme in lead poisoning. Human PBGS participates in a quaternary structure equilibrium that includes high activity octamers, low activity hexamers, and two conformations of a dimer, each of which dictates the stoichiometry of further assembly. Mutations that perturb the quaternary structure equilibrium toward the low activity hexamer are associated with ALAD porphyria, a rare but serious disease state. Photodynamic therapy of dermatologic lesions with the drug Levulan requires PBGS activity. Two distinct aims are proposed.
AIM 1 investigates whether drugs and environmental contaminants can modulate the human PBGS quaternary structure equilibrium and thus inhibit or activate enzyme activity. Libraries of approved drugs and known environmental contaminants will be evaluated by a native PAGE mobility shift assay to determine which components can shift the human PBGS quaternary structure equilibrium toward either the low or high activity states. Established methods will be used to validate hits as inhibitors or activators. Inhibitors are molecules that should be avoided in patients with lead poisoning or porphyria;activators may alleviate some symptoms of lead poisoning, serve as a therapeutic for ALAD porphyria, or as adjuvant to optimize the outcome of photodynamic therapy. Coupled with the understanding of how human allelic variation affects the equilibrium of assembly states, these studies introduce a new approach to the application of quaternary structure to personalized medicine. They provide a possible explanation for off-target side effects, and also suggest off-label therapies.
AIM 2 uses human PBGS as a model system for understanding the structural basis of half-of-the-sites reactivity (HSR). HSR is similar to allosteric regulation in that binding phenomena at one location in the protein profoundly affect the binding/reactivity behavior at a distant site in the same protein. Intersubunit communication is required for HSR and, in general, the molecular basis for the required communication is poorly understood. Our understanding of human PBGS structure and mechanism allows us to modulate the individual intersubunit interfaces, determine which is responsible for the required communication, and probe individual residues to determine the communication pathway. Quantitative Schiff base trapping will be used as a diagnostic for HSR. We have established that intersubunit interactions that occur when the active site lid is closed are required for maintenance of the active octamer. We propose that substrate/product binding, which is essential for active site lid closure, is therefore also essential for maintenance of the octamer and dictates the requirement for HSR. The proposed studies will improve our understanding of the role of alternate quaternary structure assemblies in human disease and will enhance general knowledge about intersubunit communication essential for HSR and some forms of allostery.

Public Health Relevance

Understanding protein structure-function relationships is essential to understanding the mechanism of action of myriad therapeutic agents. Investigation of porphobilinogen synthase, which participates in a newly discovered regulation mechanism involving quaternary structure dynamics, provides a novel structural basis for understanding the role of alternate protein assemblies in human disease, provides a mechanism for understanding drug side effects, and suggests new therapies. Inactivation of human PBGS is involved in the human conditions of ALAD porphyria and lead poisoning, the latter of which remains a pervasive public health problem in the United States and around the world.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003654-27
Application #
8324651
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Balshaw, David M
Project Start
1991-04-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
27
Fiscal Year
2012
Total Cost
$272,089
Indirect Cost
$116,164
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S et al. (2014) wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs. J Med Chem 57:2498-510
Jaffe, Eileen K (2013) Impact of quaternary structure dynamics on allosteric drug discovery. Curr Top Med Chem 13:55-63
Jaffe, Eileen K; Lawrence, Sarah H (2012) Allostery and the dynamic oligomerization of porphobilinogen synthase. Arch Biochem Biophys 519:144-53
Jaffe, Eileen K; Lawrence, Sarah H (2012) The morpheein model of allostery: evaluating proteins as potential morpheeins. Methods Mol Biol 796:217-31
Selwood, Trevor; Jaffe, Eileen K (2012) Dynamic dissociating homo-oligomers and the control of protein function. Arch Biochem Biophys 519:131-43
Jaffe, Eileen K; Shanmugam, Dhanasekaran; Gardberg, Anna et al. (2011) Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation. J Biol Chem 286:15298-307
Lawrence, Sarah H; Selwood, Trevor; Jaffe, Eileen K (2011) Diverse clinical compounds alter the quaternary structure and inhibit the activity of an essential enzyme. ChemMedChem 6:1067-73
Ramirez, Ursula D; Myachina, Faina; Stith, Linda et al. (2010) Docking to large allosteric binding sites on protein surfaces. Adv Exp Med Biol 680:481-8
Shanmugam, Dhanasekaran; Wu, Bo; Ramirez, Ursula et al. (2010) Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential. J Biol Chem 285:22122-31
Lawrence, Sarah H; Ramirez, Ursula D; Selwood, Trevor et al. (2009) Allosteric inhibition of human porphobilinogen synthase. J Biol Chem 284:35807-17

Showing the most recent 10 out of 45 publications