Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats
Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured thei...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2021-11, Vol.118 (46), p.1-11 |
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| creator | Lindenburg, Laurens H. Pantelejevs, Teodors Gielen, Fabrice Zuazua-Villar, Pedro Butz, Maren Rees, Eric Kaminski, Clemens F. Downs, Jessica A. Hyvönen, Marko Hollfelder, Florian |
| description | Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −22.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation. |
| doi_str_mv | 10.1073/pnas.2017708118 |
| format | Article |
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Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −22.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.2017708118</identifier><identifier>PMID: 34772801</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Amino Acid Sequence ; Binders ; Binding ; Biological Sciences ; BRCA2 protein ; BRCA2 Protein - metabolism ; Breast cancer ; Cell Line, Tumor ; Chimeras ; Crystal structure ; Humans ; Ionizing radiation ; Modularity ; Modules ; Mutation ; Protein Binding - physiology ; Rad51 Recombinase - metabolism</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2021-11, Vol.118 (46), p.1-11</ispartof><rights>Copyright National Academy of Sciences Nov 16, 2021</rights><rights>2021</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c443t-a1a7954092acd44f180d0f98625026af21390861a07ccedb3b4690834b39c0223</citedby><cites>FETCH-LOGICAL-c443t-a1a7954092acd44f180d0f98625026af21390861a07ccedb3b4690834b39c0223</cites><orcidid>0000-0001-6775-8041 ; 0000-0003-2220-6379 ; 0000-0001-8683-4070 ; 0000-0002-1367-6312</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/27093905$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/27093905$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27922,27923,53789,53791,58015,58248</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34772801$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lindenburg, Laurens H.</creatorcontrib><creatorcontrib>Pantelejevs, Teodors</creatorcontrib><creatorcontrib>Gielen, Fabrice</creatorcontrib><creatorcontrib>Zuazua-Villar, Pedro</creatorcontrib><creatorcontrib>Butz, Maren</creatorcontrib><creatorcontrib>Rees, Eric</creatorcontrib><creatorcontrib>Kaminski, Clemens F.</creatorcontrib><creatorcontrib>Downs, Jessica A.</creatorcontrib><creatorcontrib>Hyvönen, Marko</creatorcontrib><creatorcontrib>Hollfelder, Florian</creatorcontrib><title>Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −22.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.</description><subject>Amino Acid Sequence</subject><subject>Binders</subject><subject>Binding</subject><subject>Biological Sciences</subject><subject>BRCA2 protein</subject><subject>BRCA2 Protein - metabolism</subject><subject>Breast cancer</subject><subject>Cell Line, Tumor</subject><subject>Chimeras</subject><subject>Crystal structure</subject><subject>Humans</subject><subject>Ionizing radiation</subject><subject>Modularity</subject><subject>Modules</subject><subject>Mutation</subject><subject>Protein Binding - physiology</subject><subject>Rad51 Recombinase - metabolism</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkd1r2zAUxcVYWbO0z33aEOxlL26uPmxJL4MuWz-gUAjNs5BtKXGwLU-yC_3vp5AuXft0Qeenwz33IHRB4JKAYIuhN_GSAhECJCHyA5oRUCQruIKPaAZARSY55afoc4w7AFC5hE_olHEhqAQyQ-u7bgj-ydZ4dfUrJ7hs-tqGiMdt8NNmizs_Ng7H7eRc2_QbXJqYWN8nwCaxnloTmvEZe4d_rpY42MGaMZ6hE2faaM9f5hytr38_Lm-z-4ebu-XVfVZxzsbMECNUzkFRU9WcOyKhBqdkQXOghXGUMAWyIAZEVdm6ZCUv0gPjJVMVUMrm6MfBd5jKztaV7cdgWj2EpjPhWXvT6LdK32z1xj9pKagAypPB9xeD4P9MNo66a2Jl29b01k9R01yJHKAAktBv79Cdn0Kf4u0ppVIMkSdqcaCq4GMM1h2XIaD3lel9Zfq1svTj6_8Zjvy_jhLw5QDs4ujDUU8BVDpPzv4C6rWa0Q</recordid><startdate>20211116</startdate><enddate>20211116</enddate><creator>Lindenburg, Laurens H.</creator><creator>Pantelejevs, Teodors</creator><creator>Gielen, Fabrice</creator><creator>Zuazua-Villar, Pedro</creator><creator>Butz, Maren</creator><creator>Rees, Eric</creator><creator>Kaminski, Clemens F.</creator><creator>Downs, Jessica A.</creator><creator>Hyvönen, Marko</creator><creator>Hollfelder, Florian</creator><general>National Academy of Sciences</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-6775-8041</orcidid><orcidid>https://orcid.org/0000-0003-2220-6379</orcidid><orcidid>https://orcid.org/0000-0001-8683-4070</orcidid><orcidid>https://orcid.org/0000-0002-1367-6312</orcidid></search><sort><creationdate>20211116</creationdate><title>Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats</title><author>Lindenburg, Laurens H. ; 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Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −22.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>34772801</pmid><doi>10.1073/pnas.2017708118</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0001-6775-8041</orcidid><orcidid>https://orcid.org/0000-0003-2220-6379</orcidid><orcidid>https://orcid.org/0000-0001-8683-4070</orcidid><orcidid>https://orcid.org/0000-0002-1367-6312</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Binders Binding Biological Sciences BRCA2 protein BRCA2 Protein - metabolism Breast cancer Cell Line, Tumor Chimeras Crystal structure Humans Ionizing radiation Modularity Modules Mutation Protein Binding - physiology Rad51 Recombinase - metabolism |
| title | Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats |
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