Article #1: Insight into the PrPc PrPsc 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

154

136 171

PrPc

PrPsc

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 PrPc to PrPsc conversion involves increase in β-sheet content.

-Even the most pure PrPsc samples are heterogeneous.

-PrP variants in sheep have different susceptibilities to forming PrPsc. 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 PrPc 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 PrPc differs from PrPsc.-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 thisstudy. 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 PrPsc.Monoclonal Ab had to be made and Fab fragment purified from it for this purpose.

epitope-binding portion

Fig. 17.14

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 domainQuestion:

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;101:10254-10259

©2004 by National Academy of Sciences

α1

α2

α3

Your Text

154

136 171

PrPc

PrPsc

Previous NMR data

Comparison to Figure inYour Text (pg. 67)

Structural consequences of scrapie-sensitivity-related mutations.

Eghiaian F et al. PNAS 2004;101:10254-10259

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 1st….

moderate HIGH

PrP polymorphisms (in sheep):

PrPc(2): A136-H154-Q171 (AHQ)-low susceptibility PrPc(3): A136-R154-Q171 (ARQ)-moderate susceptibility PrPsc: V136-R154-Q171 (VRQ)-HIGH susceptibility

How do PrP mutations affect folding?

PrPc(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;101:10254-10259

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):

PrPc(2): A136-H154-Q171 (AHQ)-low susceptibility PrPc(3): A136-R154-Q171 (ARQ)-moderate susceptibility PrPsc: V136-R154-Q171 (VRQ)-HIGH susceptibility

How do PrP mutations affect folding?

PrPc(1): A136-R154-R171 (ARR)-RESISTANT

(PNAS 101:10254)

destabilizes

repels

moderate RESISTANT

formsH bond

Position 171 R >Q in all susceptible forms

Structural consequences of scrapie-sensitivity-related mutations.

Eghiaian F et al. PNAS 2004;101:10254-10259

©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 188-199-C terminal part of H2 (one face only) and N terminal part of H2-H3 loop

Eghiaian F et al. PNAS 2004;101:10254-10259

B)-Question:

-Experiment:

-Data Interpretation:

Quantitative Elisa assays(See next slide)

Does Fab fragment bind both PrPsc and PrPc?

HRP-conjugatedVRQ14 Ab (source of Fab)

PrP sample

non-conjugatedVRQ14 Ab?

Sandwich Elisa Assay

Serial dilution of PrP

In Competitive Elisa(lower panel)PrP incubated withserial dilutions ofFab fragment first

B)-Question:

-Experiment:

-Data Interpretation:

Does Fab fragment bind both PrPsc and PrPc?

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 188-199-which containsC 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 ofsusceptible 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 PrPsc is helix H1 and flanking non-structured S1 and S2 (S1-H1-S2)N-terminus.-PrPsc conversion likely to involvethese regions.

-Mutations in ovPrP variants lie in this region.

Update:Model of Structural Changes in N-terminus Based on Data from this Workand Other Work

Residues 90-176 form Left-handed β-helix(helix composed of β-sheets)

S1-H1-S2N-terminus

determined by Electron Crystallography

d=2.5Ao

d=12Ao