Structure of RPA 32 bound to the N‐terminus of SMARCAL 1 redefines the binding interface between RPA 32 and its interacting proteins
Si Xiea b, Yao Luc d, Jean Jakoncice, Hongzhe Sunf g, Jiang Xiac d, Chengmin Qiana b✉
- aDepartment of Biochemistry the University of Hong Kong China
- bDepartment of Biochemistry, the University of Hong Kong, China
- cDepartment of Chemistry the Chinese University of Hong Kong China
- dDepartment of Chemistry, The Chinese University of Hong Kong, China
- eBrookhaven National Laboratory Photon Science Directorate Upton NY USA
- f
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Abstract
Replication protein A subunit RPA32 contains a C-terminal domain that interacts with a variety of DNA damage response proteins including SMARCAL1, Tipin, UNG2 and XPA. We have solved the high-resolution crystal structure of RPA32 C-terminal domain (RPA32C) in complex with a 26-amino-acid peptide derived from the N-terminus of SMARCAL1 (SMARCAL1N). The RPA32C-SMARCAL1N structure reveals a 1 : 1 binding stoichiometry and displays a well-ordered binding interface. SMARCAL1N adopts a long α-helical conformation with the highly conserved 11 residues aligned on one face of the α-helix showing extensive interactions with the RPA32C domain. Extensive mutagenesis experiments were performed to corroborate the interactions observed in crystal structure. Moreover, the α1/α2 loop of the RPA32C domain undergoes a conformational rearrangement upon SMARCAL1N binding. NMR study has further confirmed that the RPA32C-SMARCAL1N interaction induces conformational changes in RPA32C. Isothermal titration calorimetry studies have also demonstrated that the conserved α-helical motif defined in the current study is required for sufficient binding of RPA32C. Taken together, our study has provided convincing structural information that redefines the common recognition pattern shared by RPA32C interacting proteins. DATABASE: The atomic coordinates of RPA32C in complex with 26-aa SMARCAL1 (SMARCAL1N) peptide have been deposited at the Protein Data Bank with accession code 4MQV. STRUCTURED DIGITAL ABSTRACT: RPA32 and SMARCAL1 bind by isothermal titration calorimetry(1, 2, 3, 4, 5, 6, 7, 8, 9) RPA32 and SMARCAL1 bind by molecular sieving (View interaction) RPA32 and SMARCAL1 bind by x-ray crystallography (View interaction) Tipin and RPA32 bind by isothermal titration calorimetry (1, 2) RPA32 and UNG2 bind by isothermal titration calorimetry (1, 2, 3) SMARCAL1 and RPA32 bind by nuclear magnetic resonance (View interaction) UNG2 and RPA32 bind by nuclear magnetic resonance (View interaction) Tipin and RPA32 bind by nuclear magnetic resonance (View interaction).