title: Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein.
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.
authors: Department of Biochemistry, ‡Department of Chemistry, §Department of Pharmacology, and ∥Center for Structural Biology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: Michael D
authors: MD
authors: Feldkamp
authors: Andreas O
authors: AO
authors: Frank
authors: J Phillip
authors: JP
authors: Kennedy
authors: James D
authors: JD
authors: Patrone
authors: Bhavatarini
authors: B
authors: Vangamudi
authors: Alex G
authors: AG
authors: Waterson
authors: Stephen W
authors: SW
authors: Fesik
authors: Walter J
authors: WJ
authors: Chazin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005454:Fluorescence Polarization; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D054730:Protein Interaction Domains and Motifs; D051716:Replication Protein A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D055672:Static Electricity
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D051716:Replication Protein A
references: Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):580-5
references: 16407120
references: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references: 21460441
references: Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15412-7
references: 16234232
references: Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523-7
references: 2833742
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Jun;69(Pt 6):592-6
references: 23722832
references: Protein Sci. 2007 Jul;16(7):1389-97
references: 17586772
references: J Nucleic Acids. 2010 Dec 06;2010:304035
references: 21188165
references: Annu Rev Biochem. 1997;66:61-92
references: 9242902
references: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
references: 11517324
references: Cancer Res. 2010 Apr 15;70(8):3189-98
references: 20395205
references: Protein Pept Lett. 2012 Jul;19(7):732-42
references: 22489782
references: Protein Sci. 2007 Aug;16(8):1569-76
references: 17656576
references: Mol Cell Biol. 2007 May;27(9):3367-77
references: 17339343
references: Bioorg Med Chem Lett. 2010 Nov 15;20(22):6587-91
references: 20870405
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: Biochem J. 2011 Jun 15;436(3):527-36
references: 21615334
references: Cell. 1998 Aug 7;94(3):399-409
references: 9708741
references: Biochemistry. 2005 Dec 6;44(48):15750-9
references: 16313178
references: J Biomol NMR. 1995 Nov;6(3):277-93
references: 8520220
references: Science. 1996 Nov 29;274(5292):1531-4
references: 8929414
references: Nucleic Acids Res. 2008 Sep;36(15):5083-92
references: 18676450
references: Anal Biochem. 2012 Feb 15;421(2):742-9
references: 22197419
references: J Mol Biol. 1996 May 31;259(1):104-12
references: 8648638
references: Nature. 1993 Sep 2;365(6441):79-82
references: 8361542
references: J Biol Chem. 2006 Dec 22;281(51):39517-33
references: 17035231
references: Nature. 1997 Jan 9;385(6612):176-81
references: 8990123
references: Mol Biol Cell. 2005 May;16(5):2372-81
references: 15743907
references: Nucleic Acids Res. 2006;34(15):4126-37
references: 16935876
references: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13827-32
references: 14605214
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references: 15572765
references: Prog Biophys Mol Biol. 2009 Nov;101(1-3):3-12
references: 20018205
references: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references: 20124702
references: Protein Sci. 2010 May;19(5):901-13
references: 20196072
references: Mol Cancer Ther. 2004 Apr;3(4):385-91
references: 15078981
references: Bioorg Med Chem. 2011 Apr 15;19(8):2589-95
references: 21459001
references: Curr Opin Drug Discov Devel. 2003 Jul;6(4):544-9
references: 12951817
references: Science. 2001 Nov 23;294(5547):1713-6
references: 11721054
references: Structure. 2004 Apr;12(4):529-35
references: 15062076
references: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):243-8
references: 21460442
references: Biochemistry. 2000 Feb 8;39(5):850-9
references: 10653628
references: FEBS Lett. 2011 Jun 6;585(11):1625-39
references: 21570395
references: J Struct Biol. 2003 Apr;142(1):88-97
references: 12718922
references: Mol Cell Biol. 2008 Dec;28(24):7345-53
references: 18936170