Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins

Abstract

Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington's disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues. The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins. Anfinsen's paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP-nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding-misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.

Figure 1

Free-energy landscape of protein folding versus misfolding. Unfolded proteins (represented in the top of the diagram) have high conformational entropy and are highly hydrated. As the proteins evolve in the funnel, the intermediate species become more structured and less hydrated. Some proteins face a bifurcation in the landscape, leading to metastable conformations, which depending on the conditions might stabilize misfolded and aggregated species.

Figure 2

Hydrostatic pressure effects in different systems. Pressure acts on proteins by water infiltration and shifts the equilibrium to smaller volumes, and may induce protein denaturation, protein dissociation, dissociation of protein−nucleic acid complexes, disassembly of aggregates, including amyloids, and formation of preamyloidogenic intermediates.

Figure 3

Energy and volume diagram of PrP misfolding. PrP^C (left) can misfold into an isoform rich in β-sheet structure capable of forming toxic and infectious aggregates (PrP^Sc) (right). The transition between the species is separated by a large energetic barrier. I and U represent intermediate and unfolded states, respectively. An adjuvant factor would lower the free-energy barrier, triggering formation of PrP^Sc. PrP^C has a larger solvent-accessible surface area than the misfolded/aggregated species, and the folding pathway also exhibits a kinetic barrier in the activation volume (inset, modified from ref (15)). The pressure-denatured states of α-rPrP (PrP^C) and β-rPrP (PrP^Sc-like) are denoted as U and U′, respectively.

Figure 4

Stabilization of p53C upon sequence-specific DNA binding and recovery of misfolded aggregated species of p53C. (A) Structure of p53C bound to DNA (PDB entry 2ABY). (B) Full-length p53 is stabilized against pressure denaturation upon DNA binding as measured by fluorescence: p53 (blue circles), consensus-bound p53 (red squares), and poly(GC)-bound p53 (green triangles). Open symbols are values after return to atmospheric pressure. (C) Cognate DNA rescues the native conformation of p53C after misfolding and aggregation. Fluorescence of wild-type p53C at atmospheric pressure (solid black line); after the first cycle of pressurization in the absence of DNA (red line); after DNA addition at atmospheric pressure (blue line); and after the second pressure cycle in the presence of DNA (green line). (D) Proposed model for p53C aggregation. Conversion of native, active p53 (blue circles) into aggregates (red squares) in the cytoplasm (upper panels). Nuclear DNA is represented in purple. Adapted from refs (49) and (51).