Structural Mechanism of Mammalian Prion Infectivity

Supattapone, Surachai; Zilm, Kurt

Abstract

Mammalian prions, which cause fatal neurodegenerative diseases in humans and livestock animals, are unorthodox infectious agents that replicate by the autocatalytic conformational change of the host prion protein (PrPC) into a pathogenic isoform (PrPSc). Taking a reductionist biochemical approach, we made the surprising discovery that endogenous cofactors such as the membrane lipid phosphatidylethanolamine (PE) are required to produce infectious PrPSc molecules. For example, autocatalytic PrPSc molecules formed with a combination of recombinant PrP and PE substrates display a specific infectivity >105-fold greater than that of similar autocatalytic PrPSc molecules formed from PrP alone. We have taken advantage of this specific information to produce, for the first time, sufficient quantities of fully infectious, isotopically labeled, recombinant prions to perform solid state (ss) NMR analysis of PrPSc structure. Here, our collaborative team proposes to use ssNMR to determine and compare the secondary structure maps of infectious and non-infectious PrPSc molecules. This work will help identify and characterize specific infectivity-associated domains as well as crucial NMR residue-peak assignments that will eventually enable structural determination of the entire PrPSc molecule. We will also use ssNMR to determine precisely the structural mechanism by which PE maintains the infectious prion conformation. Finally, we will use 15N-13C transferred-echo double resonance (TEDOR) to determine the symmetry pattern of PrPSc subunits in isotopically mixed samples of infectious recombinant prions. These data will allow us to discriminate between competing ?-solenoid and in-register ?-sheet quaternary structure models of infectious PrPSc molecules. Overall, this proposal is a critical step towards determining the full high-resolution structural determination of infectious mammalian prions as well as the structural mechanism of prion infectivity.

Public Health Relevance

Prions are unorthodox infectious agents that cause fatal brain diseases in humans and livestock animals. Unlike conventional infectious agents, prions do not contain DNA or any other forms of genetic information, and instead appear to cause disease by causing protein misfolding. The process by which prions cause protein misfolding in the brain may also be responsible for other human brain disorders associated with aging such as Alzheimer's disease and Parkinson's disease, as well as the spread of brain damage caused by physical trauma. In this proposal, taking advantage of our unique ability to produce large quantities of chemically pure infectious prions, our collaborative team will study for the first time the detailed molecular structure of an infectious prion using high- resolution techniques. By directly comparing infectious against non-infectious prion molecules, we will identify structural elements that are essential for infectivity. The results of this research will help us visualize in molecular detail the process by which proteins are induced to change their shape and cause disease. Ultimately, this structural information will enable us to develop logical methods to specifically diagnose and combat prion diseases and related disorders.