A Case StudyA Case StudyRPA: A Multi-domain, Multi-subunit ProteinRPA: A Multi-domain, Multi-subunit Protein

RPA70

RPA32

RPA14

Zn

P

Quaternary structure unknown, partial function

Delineation of domains by limited proteolysis

Binds ssDNA

Binds proteins

• DNA damage must be repaired

• Malfuction of repair leads to cancer

• Goal: Understand repair to make anticancer drug

RPA is an essential component of the NER pathwayRPA is an essential component of the NER pathway

Protein Interactions in Biology:Protein Interactions in Biology: RPA/XPA in Nucleotide Excision RepairRPA/XPA in Nucleotide Excision Repair

TFIIHXPF

XPA

XPC

XPG

RPA

TFIIH

2

The NER Complex is a Protein MachineThe NER Complex is a Protein Machine

XPF5

XPC

1

XPA

3

XPG

4

RPA

3,4,5….

1. Recognize damage

3. Locate lesion

4. Excise 5’

5. Excise 3’

2. Unwind duplex

RPA is required at multiple steps

Machines perform multiple tasks

Probing RPA/XPA InteractionsProbing RPA/XPA Interactions

Proteolysis

Elute

Mass SpecIdentification

Wash

XPAXPARPA14/32RPA14/32 Affinity Affinity

XPA1-273

FT E E FT EFT

Control *14/32 †14/32

Binding of XPA by RPA14/32Binding of XPA by RPA14/32

SDS-PAGE

* Mass Spec: all bound fragments have XPA1-98

† C-terminus of RPA32 required for binding XPA

XPA N-Terminal Domain Binds RPAXPA N-Terminal Domain Binds RPA

FT W1 W2 E E

Control 14/32 14/32

FT W1 W2 E FT W1 W2 E

XPA1-98

SDS-PAGE

Isolate the RPA32 C-terminal Domain Isolate the RPA32 C-terminal Domain to Determine its Functionto Determine its Function

17340

RPA32

RPA14

P XPAXPA

Produce RPA32 C-terminal domain (RPA32C)

RPA32C

RPA32C NMR StructureRPA32C NMR StructureThe Starting Point!The Starting Point!

C

N

Winged Helix-Loop-Helix

• Only 19 residues affected Discrete binding site

• Exchange broadening Kd > 1 M

RPA32CRPA32C + XPA 1-98

Use NMR to Define XPA Binding SiteUse NMR to Define XPA Binding Site1515N-RPA32C + Unlabeled XPAN-RPA32C + Unlabeled XPA1-981-98

15N - C- CO - - -15N - C

H

R R

H

Perturbations in NMR Spectrum Perturbations in NMR Spectrum Mapped onto RPA32C StructureMapped onto RPA32C Structure

C

N

Winged Helix-Loop-Helix

Discrete surfaceDiscrete surface

Different from HLH surface for dsDNA

RPA32C does not bind dsDNA

Use NMR to Define RPA-Binding SiteUse NMR to Define RPA-Binding Site Titration of Titration of 1515N-XPAN-XPA1-98 1-98 + RPA32C+ RPA32C

XPA1-98

XPA1-98 + RPA32C

MAAADGALPEAAALEQPAELPAS

VRASIERKRQQRALMLRQQARLAAR

PYSATAAAATGGMANVKAAPKIID

TGGGFILEEEEEEEQKIGKVVHQP

GPVM

- 15NH - C- CO -

(CH2)n- C - 15NH2

O

XPA1-98 XPA29-46

• Same residues Same binding site

• Slow exchange Kd < 1 M

XPA Peptide Induces Same Shifts in XPA Peptide Induces Same Shifts in RPA32C as Intact N-terminal DomainRPA32C as Intact N-terminal Domain

Predict Binding Sites in Other DNA Predict Binding Sites in Other DNA Damage Recognition ProteinsDamage Recognition Proteins

Patterns But Not Homology!!!Patterns But Not Homology!!!

E R K R Q R A L M L R Q A R L A A R

R I Q R N K A A A L L R L A A R

R K L R Q K Q L Q Q Q F R E R M E K

XPA29-46

UDG79-88

RAD257-274

NER

BER

RR

XPAXPA2929-46UDGUDG79-8879-88 RADRAD257-274257-274

NMR Shows Binding of DNA Damage NMR Shows Binding of DNA Damage Recognition Proteins is IdenticalRecognition Proteins is Identical

RPA Function From Structural Analysis RPA Function From Structural Analysis Regulator of DNA Repair PathwaysRegulator of DNA Repair Pathways

NER

BER

RR

RPA32RPA32

Molecular Basis for RPA32C InteractionsMolecular Basis for RPA32C InteractionsStructure of UDG Peptide Complex Structure of UDG Peptide Complex

NN

RPA32C-UDGRPA32C-UDG RPA32CRPA32C

C

Detailed Insights by Identifying Detailed Insights by Identifying Critical Interactions in the ComplexCritical Interactions in the Complex

Structure reveals why 3 different DNA damage recognition proteins bind to RPA32

How to generate specificity in drug targeting?

TFIIH

2

How Does the NER Machine Function?How Does the NER Machine Function?

XPF5

XPC

1

XPA

3

XPG

4

RPA

3,4,5….

1. Recognize damage

3. Locate lesion

4. Excise 5’

5. Excise 3’

2. Unwind duplex

RPA is required at multiple steps

Structural model for the NER machine must provide for progress through the multiple steps of NER?

TFIIH

2

Is the NER Complex Pre-formed?Is the NER Complex Pre-formed?

XPF5

XPC

1

XPA

3

XPG

4

RPA

3,4,5….

1. Recognize damage

3. Locate lesion

4. Excise 5’

5. Excise 3’

2. Unwind duplex

RPA is required at multiple steps

Progression through the multiple steps of NER byreorganization of a static complex

TFIIH

2

Is the NER Complex a Dynamic Assembly?Is the NER Complex a Dynamic Assembly?

XPF5

XPC

1

XPA

3

XPG

4

RPA

3,4,5….

1. Recognize damage

3. Locate lesion

4. Excise 5’

5. Excise 3’

2. Unwind duplex

RPA is required at multiple steps

Progression through the multiple steps of NER by dynamic asembly/disassembly of the complex

TFIIH

2

NMR is a Powerful Means to Study NMR is a Powerful Means to Study Dynamic Biomolecular SystemsDynamic Biomolecular Systems

XPF5

XPC

1

XPA

3

XPG

4

RPA

3,4,5….

• Progression by multiple short-lived interactionsProgression by multiple short-lived interactions

• Modularity facilitates dynamic assemblyModularity facilitates dynamic assembly