A Case Study RPA: 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 pathway Protein Interactions in Biology: RPA/XPA in Nucleotide Excision Repair TFIIH XPF XPA XPC XPG RPA

TFIIH 2 The NER Complex is a Protein Machine XPF 5 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 Interactions Proteolysis Elute Mass Spec Identification Wash XPA RPA14/32 Affinity Affinity

XPA FTEE E Control*14/32 † 14/  32 Binding of XPA by RPA14/32 SDS-PAGE * Mass Spec: all bound fragments have XPA 1-98 † C-terminus of RPA32 required for binding XPA

XPA N-Terminal Domain Binds RPA FT W1 W2 E E Control14/32 14/  32 FT W1 W2 E XPA 1-98 SDS-PAGE

Isolate the RPA32 C-terminal Domain to Determine its Function RPA32 RPA14 P XPA Produce RPA32 C-terminal domain (RPA32C) RPA32C

RPA32C NMR Structure The Starting Point! C N Winged Helix-Loop-Helix

Only 19 residues affected  Discrete binding site Exchange broadening  Kd > 1  M RPA32C RPA32C + XPA 1-98 Use NMR to Define XPA Binding Site 15 N-RPA32C + Unlabeled XPA N - C  - CO N - C  H RR H

Perturbations in NMR Spectrum Mapped onto RPA32C Structure C N Winged Helix-Loop-Helix  Discrete surface  Different from HLH surface for dsDNA  RPA32C does not bind dsDNA

Use NMR to Define RPA-Binding Site Titration of 15 N-XPA RPA32C XPA 1-98 XPA RPA32C QQ MAAADGALPEAAALEQPAELPASV RASIERKR Q RALMLR Q ARLAARP YSATAAAATGGMANVKAAPKIIDT GGGFILEEEEEEEQKIGKVVHQPG PVM - 15 NH - C  - CO - (CH 2 ) n - C - 15 NH 2 O

XPA 1-98 XPA Same residues  Same binding site Slow exchange  Kd < 1  M XPA Peptide Induces Same Shifts in RPA32C as Intact N-terminal Domain

Predict Binding Sites in Other DNA Damage Recognition Proteins Patterns But Not Homology!!! E R K R Q R A L M L R Q A R L A A RR I Q R N K A A A L L R L A A RR K L R Q K Q L Q Q Q F R E R M E KE R K R Q R A L M L R Q A R L A A RR I Q R N K A A A L L R L A A RR K L R Q K Q L Q Q Q F R E R M E K XPA UDG RAD NER BER RR

XPA 29 XPA UDG RAD NMR Shows Binding of DNA Damage Recognition Proteins is Identical

RPA Function From Structural Analysis Regulator of DNA Repair Pathways NER BER RR RPA32

Molecular Basis for RPA32C Interactions Structure of UDG Peptide Complex N N RPA32C-UDGRPA32C C

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?

TFIIH 2 How Does the NER Machine Function? XPF 5 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? XPF 5 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 reorganization of a static complex

TFIIH 2 Is the NER Complex a Dynamic Assembly? XPF 5 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 Dynamic Biomolecular Systems XPF 5 XPC 1 XPA 3 XPG 4 RPA 3,4,5…. Progression by multiple short-lived interactions Progression by multiple short-lived interactions Modularity facilitates dynamic assembly Modularity facilitates dynamic assembly