Nonnatural Protein-Protein Interaction-Pair Design by Key Residues Grafting
Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for...
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Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2007-03, Vol.104 (13), p.5330-5335 |
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description | Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat ${\rm PLC}\delta _{1}-{\rm PH}$ (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat ${\rm PLC}\delta _{1}-{\rm PH}$ were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of ${\rm PLC}\delta _{1}-{\rm PH}$ (ERPH1) was found to bind EPOR with high affinity $K_{D}$ of 24 nM and an ${\rm IC}_{50}$ of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT ${\rm PLC}\delta _{1}-{\rm PH}$ did not show any detectable binding under the assay conditions. The in vitro binding affinities of the ${\rm PLC}\delta _{1}-{\rm PH}$ mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein- protein interfaces. |
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In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat ${\rm PLC}\delta _{1}-{\rm PH}$ (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat ${\rm PLC}\delta _{1}-{\rm PH}$ were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of ${\rm PLC}\delta _{1}-{\rm PH}$ (ERPH1) was found to bind EPOR with high affinity $K_{D}$ of 24 nM and an ${\rm IC}_{50}$ of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT ${\rm PLC}\delta _{1}-{\rm PH}$ did not show any detectable binding under the assay conditions. The in vitro binding affinities of the ${\rm PLC}\delta _{1}-{\rm PH}$ mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein- protein interfaces.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.0606198104</identifier><identifier>PMID: 17372228</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Algorithms ; Animals ; Atoms ; Binding sites ; Biochemistry ; Biological Sciences ; Blotting, Western ; Cells ; Databases, Protein ; Epics ; Epitopes ; Gene expression ; Genetic engineering ; Humans ; Inhibitory Concentration 50 ; Kinetics ; Models, Molecular ; Mutation ; Packing density ; Protein Conformation ; Protein Interaction Mapping ; Protein Structure, Secondary ; Proteins ; Proteins - chemistry ; Rats ; Receptors ; Scaffolds ; Surface areas ; Surface Plasmon Resonance</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2007-03, Vol.104 (13), p.5330-5335</ispartof><rights>Copyright 2007 The National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Mar 27, 2007</rights><rights>2007 by The National Academy of Sciences of the USA 2007</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c594t-8df2267db3470d0d9f530597795e2dd107eac1e1f05bf8f339ae24fe64ff01903</citedby><cites>FETCH-LOGICAL-c594t-8df2267db3470d0d9f530597795e2dd107eac1e1f05bf8f339ae24fe64ff01903</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/104/13.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/25427195$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/25427195$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27923,27924,53790,53792,58016,58249</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17372228$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Liu, Sen</creatorcontrib><creatorcontrib>Liu, Shiyong</creatorcontrib><creatorcontrib>Zhu, Xiaolei</creatorcontrib><creatorcontrib>Liang, Huanhuan</creatorcontrib><creatorcontrib>Cao, Aoneng</creatorcontrib><creatorcontrib>Chang, Zhijie</creatorcontrib><creatorcontrib>Lai, Luhua</creatorcontrib><title>Nonnatural Protein-Protein Interaction-Pair Design by Key Residues Grafting</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat ${\rm PLC}\delta _{1}-{\rm PH}$ (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat ${\rm PLC}\delta _{1}-{\rm PH}$ were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of ${\rm PLC}\delta _{1}-{\rm PH}$ (ERPH1) was found to bind EPOR with high affinity $K_{D}$ of 24 nM and an ${\rm IC}_{50}$ of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT ${\rm PLC}\delta _{1}-{\rm PH}$ did not show any detectable binding under the assay conditions. The in vitro binding affinities of the ${\rm PLC}\delta _{1}-{\rm PH}$ mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein- protein interfaces.</description><subject>Algorithms</subject><subject>Animals</subject><subject>Atoms</subject><subject>Binding sites</subject><subject>Biochemistry</subject><subject>Biological Sciences</subject><subject>Blotting, Western</subject><subject>Cells</subject><subject>Databases, Protein</subject><subject>Epics</subject><subject>Epitopes</subject><subject>Gene expression</subject><subject>Genetic engineering</subject><subject>Humans</subject><subject>Inhibitory Concentration 50</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutation</subject><subject>Packing density</subject><subject>Protein Conformation</subject><subject>Protein Interaction Mapping</subject><subject>Protein Structure, Secondary</subject><subject>Proteins</subject><subject>Proteins - chemistry</subject><subject>Rats</subject><subject>Receptors</subject><subject>Scaffolds</subject><subject>Surface areas</subject><subject>Surface Plasmon Resonance</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1vEzEQxS0EoqFw5gSsOCAu246_1vYFCRUoVStACM6Ws2sHRxs7tb2I_Pc4yqoBDpxGM_7N8zw9hJ5iOMMg6Pk2mHwGHXRYSQzsHlpgULjtmIL7aAFARCsZYSfoUc5rAFBcwkN0ggUVhBC5QNefYgimTMmMzZcUi_WhnWtzFYpNpi8-1pnxqXlns1-FZrlrru2u-Vq7YbK5uUzGFR9Wj9EDZ8Zsn8z1FH3_8P7bxcf25vPl1cXbm7bnipVWDo6QTgxLygQMMCjHKXAlhOKWDEP1ZU2PLXbAl046SpWxhDnbMecAK6Cn6M1BdzstN3bobSj1fL1NfmPSTkfj9d8vwf_Qq_hTY0kl63gVeDULpHhbHRS98bm342iCjVPWWFWoo_ufXv4DruOUQjWnCWDKFZWqQucHqE8x52Td3SUY9D4lvU9JH1OqG8__NHDk51gq8HoG9ptHOaYx1ZxS0G4ax2J_lYq--D9aiWcHYp1LTHcI4YwIrDj9DWpOsBQ</recordid><startdate>20070327</startdate><enddate>20070327</enddate><creator>Liu, Sen</creator><creator>Liu, Shiyong</creator><creator>Zhu, Xiaolei</creator><creator>Liang, Huanhuan</creator><creator>Cao, Aoneng</creator><creator>Chang, Zhijie</creator><creator>Lai, Luhua</creator><general>National Academy of Sciences</general><general>National Acad 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>7QO</scope><scope>5PM</scope></search><sort><creationdate>20070327</creationdate><title>Nonnatural Protein-Protein Interaction-Pair Design by Key Residues Grafting</title><author>Liu, Sen ; Liu, Shiyong ; Zhu, Xiaolei ; Liang, Huanhuan ; Cao, Aoneng ; Chang, Zhijie ; Lai, Luhua</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c594t-8df2267db3470d0d9f530597795e2dd107eac1e1f05bf8f339ae24fe64ff01903</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Algorithms</topic><topic>Animals</topic><topic>Atoms</topic><topic>Binding sites</topic><topic>Biochemistry</topic><topic>Biological Sciences</topic><topic>Blotting, Western</topic><topic>Cells</topic><topic>Databases, Protein</topic><topic>Epics</topic><topic>Epitopes</topic><topic>Gene expression</topic><topic>Genetic engineering</topic><topic>Humans</topic><topic>Inhibitory Concentration 50</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutation</topic><topic>Packing density</topic><topic>Protein Conformation</topic><topic>Protein Interaction Mapping</topic><topic>Protein Structure, Secondary</topic><topic>Proteins</topic><topic>Proteins - chemistry</topic><topic>Rats</topic><topic>Receptors</topic><topic>Scaffolds</topic><topic>Surface areas</topic><topic>Surface Plasmon Resonance</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Sen</creatorcontrib><creatorcontrib>Liu, Shiyong</creatorcontrib><creatorcontrib>Zhu, Xiaolei</creatorcontrib><creatorcontrib>Liang, Huanhuan</creatorcontrib><creatorcontrib>Cao, Aoneng</creatorcontrib><creatorcontrib>Chang, Zhijie</creatorcontrib><creatorcontrib>Lai, Luhua</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Sen</au><au>Liu, Shiyong</au><au>Zhu, Xiaolei</au><au>Liang, Huanhuan</au><au>Cao, Aoneng</au><au>Chang, Zhijie</au><au>Lai, Luhua</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nonnatural Protein-Protein Interaction-Pair Design by Key Residues Grafting</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2007-03-27</date><risdate>2007</risdate><volume>104</volume><issue>13</issue><spage>5330</spage><epage>5335</epage><pages>5330-5335</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat ${\rm PLC}\delta _{1}-{\rm PH}$ (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat ${\rm PLC}\delta _{1}-{\rm PH}$ were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of ${\rm PLC}\delta _{1}-{\rm PH}$ (ERPH1) was found to bind EPOR with high affinity $K_{D}$ of 24 nM and an ${\rm IC}_{50}$ of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT ${\rm PLC}\delta _{1}-{\rm PH}$ did not show any detectable binding under the assay conditions. The in vitro binding affinities of the ${\rm PLC}\delta _{1}-{\rm PH}$ mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein- protein interfaces.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>17372228</pmid><doi>10.1073/pnas.0606198104</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Algorithms Animals Atoms Binding sites Biochemistry Biological Sciences Blotting, Western Cells Databases, Protein Epics Epitopes Gene expression Genetic engineering Humans Inhibitory Concentration 50 Kinetics Models, Molecular Mutation Packing density Protein Conformation Protein Interaction Mapping Protein Structure, Secondary Proteins Proteins - chemistry Rats Receptors Scaffolds Surface areas Surface Plasmon Resonance |
title | Nonnatural Protein-Protein Interaction-Pair Design by Key Residues Grafting |
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