4IPD

Structure of the N-terminal domain of RPA70, E100R mutant

4IPD の概要

• エントリーDOI: 10.2210/pdb4ipd/pdb
• 関連するPDBエントリー: 4IPC 4IPG 4IPH
• 分子名称: Replication protein A 70 kDa DNA-binding subunit (2 entities in total)
• 機能のキーワード: ob-fold, protein binding
• 由来する生物種: Homo sapiens (human)
• 細胞内の位置: Nucleus: P27694
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 13497.73

構造登録者

Feldkamp, M.D.,Frank, A.O.,Vangamudi, B.,Fesik, S.W.,Chazin, W.J. (登録日: 2013-01-09, 公開日: 2013-09-11, 最終更新日: 2023-09-20)

主引用文献

Feldkamp, M.D.,Frank, A.O.,Kennedy, J.P.,Patrone, J.D.,Vangamudi, B.,Waterson, A.G.,Fesik, S.W.,Chazin, W.J.
Surface Reengineering of RPA70N Enables Cocrystallization with an Inhibitor of the Replication Protein A Interaction Motif of ATR Interacting Protein.
Biochemistry, 52:6515-6524, 2013

PubMed Abstract: Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

PubMed: 23962067
DOI: 10.1021/bi400542z

実験手法

X-RAY DIFFRACTION (1.51 Å)