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Structure of RPA 32 bound to the N‐terminus of SMARCAL 1 redefines the binding interface between RPA 32 and its interacting proteins
Author(s) -
Xie Si,
Lu Yao,
Jakoncic Jean,
Sun Hongzhe,
Xia Jiang,
Qian Chengmin
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12867
Subject(s) - isothermal titration calorimetry , replication protein a , crystallography , protein structure , biophysics , protein–protein interaction , chemistry , biochemistry , dna binding protein , biology , transcription factor , gene
Replication protein A subunit RPA 32 contains a C‐terminal domain that interacts with a variety of DNA damage response proteins including SMARCAL 1, Tipin, UNG 2 and XPA . We have solved the high‐resolution crystal structure of RPA 32 C‐terminal domain ( RPA 32C) in complex with a 26‐amino‐acid peptide derived from the N‐terminus of SMARCAL 1 ( SMARCAL 1N). The RPA 32C– SMARCAL 1N structure reveals a 1 : 1 binding stoichiometry and displays a well‐ordered binding interface. SMARCAL 1N 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 RPA 32C domain. Extensive mutagenesis experiments were performed to corroborate the interactions observed in crystal structure. Moreover, the α1/α2 loop of the RPA 32C domain undergoes a conformational rearrangement upon SMARCAL 1N binding. NMR study has further confirmed that the RPA 32C– SMARCAL 1N interaction induces conformational changes in RPA 32C. Isothermal titration calorimetry studies have also demonstrated that the conserved α‐helical motif defined in the current study is required for sufficient binding of RPA 32C. Taken together, our study has provided convincing structural information that redefines the common recognition pattern shared by RPA 32C 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 abstractRPA32 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 )