Final Project

I. Abstract

Prions are misfolded proteins that exhibit ‘infectious’-like behaviour and are the root cause for multiple prion protein associated neurodiseases: Creutzfeldt–Jakob Disease, Kuru (not exclusively in humans) and Scrapie and Bovine spongiform encephalopathy (BSE, mad-cow disease) characteristically affecting ovines and bovines respectively.

In humans, the gene PrP^C encodes for the premature version of the cellular prion protein (253-a.a. long) found within neuroglia and neurons, its function and role remain ambiguous and have not been described fully (or so it is believed). After post-translational modification in the endoplasmic reticulum, the precursor protein loses its N-terminal signal peptide (1-22) and C-terminal signal peptide (232-253) and carries on as a mature, functional PrP^C surface glycoprotein that is 208-209 residues long. Faulty folding or interaction with other midfolded prion proteins can lead the native cellular variant of the protein to display a different structure and, thus, behaviour: resulting isoform is called PrP^Sc (scrapie-associated prion protein).

Present study explores how the native variant of the prion protein in humans behaves under different parameters set in-silico. Furthermore, a proposal for assessing prion diseases using binding peptides is described and showcased on PrP^C.

II. Introduction

III. Structure

*EXPLAIN ‘GLYCO-’ PART OF THE PROTEIN: glycosylphosphatidylinositol; function within cell;

For the means of this in-silico workthrough, the chosen structure for the PrP^C protein is the RCSB deposit under the name of 1QLX, obtained via NMR.

1QLX publication abstract

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90 –230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered ‘‘tail.’’ The globular domain contains three a-helices comprising the residues 144–154, 173–194, and 200 –228 and a short anti-parallelb-sheet comprising the residues 128 –131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219 –228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219 –226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions

The human PrP protein, including 2 C-terminal fragements NMR screeing resulted in the deposit 1QLX.

Important aspects of the globular domain (125-228):

three α-helices (144-154; 173-194; 200-228)
one short anti-parallel β-sheet (128-131/ 161-164)
three disordered peptides (167-171 - a loop-like structure; 187-194; 219-228)
Between the Cys179 and Cys214 residues, a stronger intramolecular force (disulfide bond) bridges helix 2 and helix 3; stabilising the native globular architecture, mechanical integrity and hydrophobic core of the monomeric protein.

Amino acid asparagine on position 181 is documented to carry a sugar chain in real cells, thus the binder would be unable to contact it.

GROMACS analysis

with the small and flat RMSD and Radius of gyration graphics, the structure can be interpreted as compact and not really seeming to “drift away” from the starting shape;
the parts that wiggle the most: the loop around 167-172 matches what the original 1QLX paper reported;

Adaptive Poisson-Boltzmann Solver in PyMol

A good binder should be able to complementary attach to electronegative and/or electropositive regions of the protein.

ConSurf analysis

IV. Binding peptide approach

V. Binder generation

Asp178 and Gln212 are both positions of disease-linked mutations.

Batch #1 - BoltzGen

1. Try one

Target residues: all from helix 2 (173-194)

Best ipTM (.443), best min PAE: spec_19_4. RMSD = 2.01

95 residues: SVTVVASGGGLVQAGGTLRVSCAASGAGVVAVSAGVRPPGSDRLLGGGVVVGKASGTVEIKDLEPADSGVVQCEAAVAGSDTLARGDGPQVTVLP

2. Try two

Target residues: solvent exposed parts from helix 2 plus some from helix 3 Filter RMSD stood within 1.2-1.9A for every speculative design, all structures should hypothetically fold accordingly.

Best ipTM (.362), best min PAE: spec_00_0. RMSD = 1.372, 2 H bonds, 0% β-sheet

49 residues: EEAKRYAIKLFVKKIEEGKPLTKRTLEELIKLAEELGDKEALEVFKKAL

3. Try three

Target residues: none choosen; let BoltzGen choose Filter RMSD stood within 1.2-1.9A for every speculative design, all structures should hypothetically fold accordingly.

Best ipTM (.460), best min PAE: spec_49_2. RMSD = 14.111, 5 H bonds, 0% α-helix

91 residues: ATASNPTITATGDKATASGTCDAPGAARTVVSLLNAETGECLGIGESDGTTASAEGTGAKAGDTVAAGVSALDAEGNILASGGGTATATPA

ipTM (.372), second best min PAE: spec_49_3. RMSD = , 12 H bonds

91 residues: ASRTCTTKSGDKLTCTASGTATDVTVTVTGEIAETSTSPSRPYTSPATGQTGSAATAVAKSKKSGEIVVCTGIVYADGTITVSCYLNGEAV