Structure of RPA32 bound to the N‐terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins. (1st July 2014)

• Record Type: Journal Article
• Authors: Xie, Si; Lu, Yao; Jakoncic, Jean; Sun, Hongzhe; Xia, Jiang; Qian, Chengmin
• Is Part Of: FEBS journal. Volume 281:Number 15(2014)
• Page Start: 3382
• Page End: 3396
• Publication Date: 2014-07-01
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12867
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12867-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs12867-sec-0001" sec-type="section"> <p>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.</p> </sec> <sec id="febs12867-sec-0002" sec-type="section"> <title>Database</title> <p>The atomic coordinates of RPA32C in complex with 26‐aa SMARCAL1 (SMARCAL1N) peptide have been deposited at the Protein Data Bank with accession code <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4MQV" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4MQV</ext-link>.</p> </sec> <sec id="febs12867-sec-0003" sec-type="section"> <title>Structured digital abstract</title> <p> <list id="febs12867-list-0001" list-type="bullet"> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9NZC9" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">SMARCAL1</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0065" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">isothermal titration calorimetry</ext-link>(<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546528" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546541" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546441" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546452" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546463" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">5</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546476" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">6</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546489" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">7</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546515" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">8</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546502" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">9</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9NZC9" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">SMARCAL1</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">molecular sieving</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546692" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9NZC9" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">SMARCAL1</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">x-ray crystallography</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546405" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9BVW5" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Tipin</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0065" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">isothermal titration calorimetry</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546608" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546593" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">2</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P13051" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">UNG2</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0065" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">isothermal titration calorimetry</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546554" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546567" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546580" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9NZC9" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">SMARCAL1</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0077" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">nuclear magnetic resonance</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546424" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P13051" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">UNG2</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0077" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">nuclear magnetic resonance</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546637" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </list-item> <list-item> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q9BVW5" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Tipin</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P15927" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">RPA32</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0077" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">nuclear magnetic resonance</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9546671" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </list-item> </list> </p> </sec> </abstract>