Structure of prion β‐oligomers as determined by short‐distance crosslinking constraint‐guided discrete molecular dynamics simulations

Abstract

The conversion of the native monomeric cellular prion protein (PrPC) into an aggregated pathological β‐oligomeric form (PrPβ) and an infectious form (PrPSc) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational changes involved in the conversion are still unknown. We applied mass spectrometry combined with chemical crosslinking, hydrogen/deuterium exchange, limited proteolysis, and surface modification for the differential characterization of the native and the urea+acid‐converted prion β‐oligomer structures to obtain insights into the mechanisms of conversion and aggregation. For the determination of the structure of the monomer and the dimer unit of the β‐oligomer, we applied a recently‐developed approach for de novo protein structure determination which is based on the incorporation of zero‐length and short‐distance crosslinking data as intra‐ and inter‐protein constraints in discrete molecular dynamics simulations (CL‐DMD). Based on all of the structural‐proteomics experimental data and the computationally predicted structures of the monomer units, we propose the potential mode of assembly of the β‐oligomer. The proposed β‐oligomer assembly provides a clue on the β‐sheet nucleation site, and how template‐based conversion of the native prion molecule occurs, growth of the prion aggregates, and maturation into fibrils may occur.

Protein Chemistry Combined with Mass Spectrometry for Protein Structure Determination

“…This allowed us to find a set of methionine and tryptophan residues that differed in the degree of modification between two different conformational states of the prion protein . This data was complementary to and in good agreement with the results from our PCASS amine-reactive reagent modification experiments …”

“…This approach is especially useful for comparing two different states of the same protein, and this comparison can reveal changes in the protection of specific residues that result from changes in conformation and/or complex formation. Noteworthy applications of the approach, as in case with limited proteolysis, are the characterization of folding intermediates − and the determination of protein interaction interfaces and of antibody epitopes for protein antigens, in particular. ,, We used this approach to study the conformational changes involved in the misfolding of the prion, α-synuclein, and tau proteins and for the characterization of the conformational changes that occur upon ligand-induced disorder-to-order protein structure transitions. ,− In a differential modification experiment, surface-modification experiments are performed in parallel for two states of the protein, and differences in the reactivities of specific amino acid residues, reflecting differences in the degree of protection or exposure, can be detected by mass spectrometry. While label-free quantitation can give an estimate of the relative amounts of modification under the two conditions, isotopically coded modification reagentswhich behave identically during the modification reaction and during mass spectrometric analysiscan provide a more accurate estimate of any changes in protection.…”

“…31 This data was complementary to and in good agreement with the results from our PCASS amine-reactive reagent modification experiments. 26 The use of functional group-specific modification reagents limits the modification reaction to a subset of amino acid residues on the surface of the protein. Thus, the use of nonselective photoreactive modification reagents, which potentially can modify any amino acid residue in the protein, would be very attractive.…”

Molecular Insights into the Misfolding and Dimerization Dynamics of the Full-Length α-Synuclein from Atomistic Discrete Molecular Dynamics Simulations

“…The solvation free energy of a protein molecule is a sum of group contributions, which are determined from values for small model compounds . The accuracy of the Medusa force field with the EEF1 implicit solvent model has been well benchmarked in protein folding and aggregation. ,, With significantly enhanced sampling efficiency and rapid computational speed, DMD simulations have been widely used in studying protein folding, amyloid aggregation, and small molecule/nanoparticle–protein interactions by both our group ,,, and others. ,− The units of mass, time, length, and energy used in our DMD simulations with an implicit water model are 1 Da, ∼50 fs, 1 Å, and 1 kcal/mol, respectively. The DMD program is available via Molecules in Action, LLC ().…”

Oligomeropathies, inflammation and prion protein binding

“…Furthermore, although the memory dysfunction was not influenced by PrP c , surface plasmon resonance enabled us to confirm a high-affinity interaction between AβOs and PrP c ( Balducci et al, 2010 ). Since these initial investigations ample data has accumulated supporting one or the other point of view ( Calella et al, 2010 ; Kessels et al, 2010 ; Cissé et al, 2011 ; Freir et al, 2011 ; Resenberger et al, 2012 ; Rushworth et al, 2013 ; Serpa et al, 2021 ).…”