Article #1: Insight into the PrP c PrP sc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants -What was known before the research was done? -What new question is the research addressing? -Were any special reagents or techniques needed for the research? -In each figure, what question is being addressed, how was it addressed, how were the data interpreted? -What new information did the research provide? Are there any caveats? What needs to be done next?

What was known before the research was done? Your Text PrP c PrP sc Previous NMR data -Prion diseases are neurodegenerative diseases that can be sporadic, heritable or acquired through an infectious agent. -Solution studies: NMR CD, etc. show the PrP c to PrP sc conversion involves increase in β-sheet content. -Even the most pure PrP sc samples are heterogeneous. -PrP variants in sheep have different susceptibilities to forming PrP sc. Initial NMR studies of these indicate loss of protein stability in both resistant and highly susceptible alleles. (Paradox) -X ray crystallography of recombinant form of human PrP c suggests it forms a dimer involving a helix 3 swap between monomers

What new question is the research addressing? -Still lack high resolution structural data for how PrP c differs from PrP sc. -Which regions of PrP are involved in the conversion, which are not? -How do naturally occurring mutations affect PrP structure Were any special reagents or techniques needed for the research? -PrP crystals for X-ray crystallography. -These have been difficult to make. Recombinant proteins lacking flexible unstructured regions, which may interfere with crystal formation, used in this study. This strategy also overcomes uncertainty associated with heterogeneity of samples from brain tissue. Recombinant proteins had to be expressed and purified from bacteria. -Fab fragment may aid in crystal formation by burying surfaces that interfere with crystal formation. These can also be used to id conserved regions in PrP sc. Monoclonal Ab had to be made and Fab fragment purified from it for this purpose.

epitope-binding portion Fig

In each figure, what question is being addressed, how was it addressed, how were the data interpreted? Fig 1: Structure of the ovPrP C-terminal domain Question: How Addressed?: Data Interpretation: 1)Where are mutations located in ovPrP? 2)How well does ovPrP-Fab complex structure overlap previously determined structure for huPrP? X-ray crystallography A) mutations located in helix H1 and flanking spacers (S1 and S2) B) rmsd (root mean square deviation) similar to those of previous NMR studies and larger than uncertainty associated w/ this study, indicating Ab Fab does not induce major changes in structure.

Eghiaian F et al. PNAS 2004;101: ©2004 by National Academy of Sciences α1α1 α2α2 α3α3 Your Text PrP c PrP sc Previous NMR data Comparison to Figure in Your Text (pg. 67)

Structural consequences of scrapie-sensitivity-related mutations. Eghiaian F et al. PNAS 2004;101: resistant HIGH MEDIUM Fig. 2 -Question: -Experiment: -Data Interpretation: How do mutations in ovPrP variants affect monomer structure? X-ray crystallography, side-by-side comparison MEDIUM ARQ vs VRQ 1 st ….

moderate HIGH PrP polymorphisms (in sheep): PrP c(2) : A136-H154-Q171 (AHQ)-low susceptibility PrP c(3) : A136-R154-Q171 (ARQ)-moderate susceptibility PrP sc : V136-R154-Q171 (VRQ)-HIGH susceptibility How do PrP mutations affect folding? PrP c(1) : A136-R154-R171 (ARR)-RESISTANT (PNAS 101:10254) H bond stabilizes Position 136 A > V makes highly susceptible

Structural consequences of scrapie-sensitivity-related mutations. Eghiaian F et al. PNAS 2004;101: resistant HIGH MEDIUM Fig. 2 -Question: -Experiment: -Data Interpretation: How do mutations in ovPrP variants affect monomer structure? X-ray crystallography, side-by-side comparison ARQ (moderate) vs VRQ (high): A to V substitution at 136 pushes N162 closer to R139, allowing H bond that stabilizes VRQ relative to ARQ MEDIUM Now ARQ vs ARR…

moderate HIGH PrP polymorphisms (in sheep): PrP c(2) : A136-H154-Q171 (AHQ)-low susceptibility PrP c(3) : A136-R154-Q171 (ARQ)-moderate susceptibility PrP sc : V136-R154-Q171 (VRQ)-HIGH susceptibility How do PrP mutations affect folding? PrP c(1) : A136-R154-R171 (ARR)-RESISTANT (PNAS 101:10254) destabilizes repels moderate RESISTANT forms H bond Position 171 R >Q in all susceptible forms

Structural consequences of scrapie-sensitivity-related mutations. Eghiaian F et al. PNAS 2004;101: ©2004 by National Academy of Sciences resistant HIGH MEDIUM Fig. 2 -Question: -Experiment: -Data Interpretation: How do mutations in ovPrP variants affect monomer structure? X-ray crystallography, side-by-side comparison ARQ (moderate) vs VRQ (high): A to V substitution at 136 pushes N162 closer to R139, allowing H bond that stabilizes VRQ relative to ARQ MEDIUM ARQ (moderate) vs ARR (resistant): Q to R substitution at 171 replaces H bond between Q171 and R167 with electrostatic repulsion that destabilizes ARQ relative to ARR

The epitope of the antibody is conserved in OvPrPC and OvPrPSc. Fig. 3 A) -Question: -Experiment: -Data Interpretation: Where does Fab fragment bind ovPrP? X-ray crystallography Residues C terminal part of H2 (one face only) and N terminal part of H2-H3 loop Eghiaian F et al. PNAS 2004;101:

B) -Question: -Experiment: -Data Interpretation: Quantitative Elisa assays (See next slide) Does Fab fragment bind both PrP sc and PrP c ?

HRP-conjugated VRQ14 Ab (source of Fab) PrP sample non-conjugated VRQ14 Ab? Sandwich Elisa Assay Serial dilution of PrP In Competitive Elisa (lower panel) PrP incubated with serial dilutions of Fab fragment first

B) -Question: -Experiment: -Data Interpretation: Does Fab fragment bind both PrPs c and PrP c ? Quantitative Elisa assays (See next slide) Upper panel Linear binding was observed to both rec PrPc, endogenous PrPc from uninfected animals and ProtK-digested PrPc + PrPsc (PrPsc). (Lower signal seen for ProtK-digested PrPc + PrPsc (PrPsc) explained by lower [PrPsc] relative to [PrPc] Conclusion: Fab epitope conserved in PrPsc (Residues which contains C terminal part of H2 (one face only) and N terminal part of H2-H3 loop). Lower panel Linear competition by Fab fragment to each PrP sample displaying binding above.

©2004 by National Academy of Sciences PrPsc-specific epitopes epitopes recognized in both PrPc and PrPsc -What new information did the research provide? -Propose: Additional H bonds in the S1-H1-S2 region of susceptible variants support β-sheet extension through H1 helix. -Perturbation of H bonds in this region in resistant variant inhibit β-sheet extension. -Data from this & other Ab epitope- binding studies (summarized in Fig.4) show that only region not conserved in PrP sc is helix H1 and flanking non-structured S1 and S2 (S1-H1-S2) N-terminus. -PrP sc conversion likely to involve these regions. -Mutations in ovPrP variants lie in this region.

Update: Model of Structural Changes in N-terminus Based on Data from this Work and Other Work Residues form Left-handed β-helix (helix composed of β-sheets) S1-H1-S2 N-terminus determined by Electron Crystallography d=2.5A o d=12A o