article #1: insight into the prp c prp sc conversion from the structures of antibody-bound ovine...
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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