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A Case Study
RPA: A Multi-domain, Multi-subunit Protein
RPA70
Zn
RPA32
P
Binds ssDNA
Binds proteins
RPA14
 Quaternary structure unknown, partial function
Delineation of domains by limited proteolysis
Protein Interactions in Biology:
RPA/XPA in Nucleotide Excision Repair
• DNA damage must be
repaired
XPC
XPF
XPA
TFIIH
XPG
RPA
• Malfuction of repair leads
to cancer
• Goal: Understand repair to
make anticancer drug
RPA is an essential component of the NER pathway
The NER Complex is a Protein Machine
1. Recognize damage
2. Unwind duplex
1
3
5
3. Locate lesion
XPC
2
XPF
XPA
TFIIH
4. Excise 5’
XPG
4
5. Excise 3’
RPA
RPA is required at multiple steps
3,4,5….
Machines perform multiple tasks
Probing RPA/XPA Interactions
Proteolysis
XPA
RPA14/32
Affinity
Wash
Elute
Mass Spec
Identification
Binding of XPA by RPA14/32
Control *14/32
FT E
XPA1-273
FT E
†14/D32
FT E
* Mass Spec:
all bound
fragments
have XPA1-98
† C-terminus
of RPA32
required for
binding XPA
SDS-PAGE
XPA N-Terminal Domain Binds RPA
Control
14/32
14/D32
XPA1-98
SDS-PAGE
FT W1 W2 E
FT W1 W2 E
FT W1 W2
E E
Isolate the RPA32 C-terminal Domain to
Determine its Function
40
RPA32
173
P
XPA
RPA14
RPA32C
Produce RPA32 C-terminal domain (RPA32C)
RPA32C NMR Structure
The Starting Point!
C
N
Winged Helix-Loop-Helix
Use NMR to Define XPA Binding Site
15N-RPA32C
+ Unlabeled XPA1-98
R
15
R
N - Ca- CO - - -15N - Ca
H
H
• Only 19 residues affected
 Discrete binding site
RPA32C
RPA32C + XPA 1-98
• Exchange broadening
 Kd > 1 mM
Perturbations in NMR Spectrum
Mapped onto RPA32C Structure
Winged Helix-Loop-Helix
 Discrete surface
C
 Different from HLH
surface for dsDNA
 RPA32C does not
bind dsDNA
N
Use NMR to Define RPA-Binding Site
Titration of 15N-XPA1-98 + RPA32C
O
(CH2)n- C - 15NH2
- 15NH - Ca- CO MAAADGALPEAAALEQPAELPASV
RASIERKRQRALMLRQARLAARP
XPA1-98
XPA1-98 + RPA32C
YSATAAAATGGMANVKAAPKIIDT
GGGFILEEEEEEEQKIGKVVHQPG
PVM
XPA Peptide Induces Same Shifts in
RPA32C as Intact N-terminal Domain
• Same residues
 Same binding site
• Slow exchange
 Kd < 1 mM
XPA1-98
XPA29-46
Predict Binding Sites in Other DNA
Damage Recognition Proteins
NER
XPA29-46
ERKRQRALMLRQARLAAR
BER
UDG79-88
RIQRNKAAALLRLAAR
RAD257-274
RKLRQKQLQQQFRERMEK
RR
Patterns But Not Homology!!!
NMR Shows Binding of DNA Damage
Recognition Proteins is Identical
XPA29-46
UDG79-88
RAD257-274
RPA Function From Structural Analysis
Regulator of DNA Repair Pathways
RPA32
NER
BER
RR
Molecular Basis for RPA32C Interactions
Structure of UDG Peptide Complex
C
N
RPA32C-UDG
N
RPA32C
Detailed Insights by Identifying
Critical Interactions in the Complex
Structure reveals
why 3 different DNA
damage recognition
proteins bind to
RPA32
 How to generate
specificity in drug
targeting?
How Does the NER Machine Function?
1. Recognize damage
2. Unwind duplex
1
3
5
3. Locate lesion
XPC
XPF
XPA
TFIIH
4. Excise 5’
5. Excise 3’
RPA is required at multiple steps
2
XPG
4
RPA
3,4,5….
Structural model for the NER machine must provide
for progress through the multiple steps of NER?
Is the NER Complex Pre-formed?
1. Recognize damage
2. Unwind duplex
1
3
5
3. Locate lesion
XPC
XPF
XPA
TFIIH
4. Excise 5’
5. Excise 3’
RPA is required at multiple steps
2
XPG
4
RPA
3,4,5….
Progression through the multiple steps of NER by
reorganization of a static complex
Is the NER Complex a Dynamic Assembly?
1. Recognize damage
2. Unwind duplex
1
3
5
3. Locate lesion
XPC
XPF
XPA
TFIIH
4. Excise 5’
5. Excise 3’
RPA is required at multiple steps
2
XPG
4
RPA
3,4,5….
Progression through the multiple steps of NER by
dynamic asembly/disassembly of the complex
NMR is a Powerful Means to Study
Dynamic Biomolecular Systems
1
3
5
XPC
2
XPF
XPA
TFIIH
XPG
4
RPA
3,4,5….
• Progression by multiple short-lived interactions
• Modularity facilitates dynamic assembly