The actin binding site of thymosin beta 4 mapped by mutational analysis
We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we...
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| Veröffentlicht in: | The EMBO journal 1996-01, Vol.15 (2), p.201-210 |
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| creator | Van Troys, M. Dewitte, D. Goethals, M. Carlier, M. F. Vandekerckhove, J. Ampe, C. |
| description | We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross‐linking, complex formation in native gels and actin‐sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N‐terminal part needs to adopt an alpha‐helix for actin binding and interacts through a patch of hydrophobic residues (6M‐I‐F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin. |
| doi_str_mv | 10.1002/j.1460-2075.1996.tb00350.x |
| format | Article |
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Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.1002/j.1460-2075.1996.tb00350.x</identifier><identifier>PMID: 8617195</identifier><language>eng</language><publisher>England</publisher><subject>Actins - chemistry ; Actins - isolation & purification ; Actins - metabolism ; Amino Acid Sequence ; Animals ; Binding Sites ; Binding, Competitive ; Blood Proteins - chemistry ; Calcium-Binding Proteins - chemistry ; Carrier Proteins - chemistry ; Chickens ; Circular Dichroism ; Conserved Sequence ; Cross-Linking Reagents ; Genetic Variation ; Humans ; Kinetics ; Membrane Proteins - chemistry ; Microfilament Proteins - chemistry ; Molecular Sequence Data ; Muscle, Skeletal - metabolism ; Mutagenesis, Site-Directed ; Peptide Fragments - chemistry ; Phosphoproteins ; Protein Structure, Secondary ; Rabbits ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sequence Homology, Amino Acid ; Thymosin - chemistry ; Thymosin - isolation & purification ; Thymosin - metabolism</subject><ispartof>The EMBO journal, 1996-01, Vol.15 (2), p.201-210</ispartof><rights>1996 European Molecular Biology Organization</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5070-e692f0072da6761faf94b8e068aaf160fd4bb779895c5fcf94e97139f85ad6093</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC449934/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC449934/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,725,778,782,883,27907,27908,53774,53776</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8617195$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Van Troys, M.</creatorcontrib><creatorcontrib>Dewitte, D.</creatorcontrib><creatorcontrib>Goethals, M.</creatorcontrib><creatorcontrib>Carlier, M. F.</creatorcontrib><creatorcontrib>Vandekerckhove, J.</creatorcontrib><creatorcontrib>Ampe, C.</creatorcontrib><title>The actin binding site of thymosin beta 4 mapped by mutational analysis</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><description>We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross‐linking, complex formation in native gels and actin‐sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N‐terminal part needs to adopt an alpha‐helix for actin binding and interacts through a patch of hydrophobic residues (6M‐I‐F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin.</description><subject>Actins - chemistry</subject><subject>Actins - isolation & purification</subject><subject>Actins - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Binding, Competitive</subject><subject>Blood Proteins - chemistry</subject><subject>Calcium-Binding Proteins - chemistry</subject><subject>Carrier Proteins - chemistry</subject><subject>Chickens</subject><subject>Circular Dichroism</subject><subject>Conserved Sequence</subject><subject>Cross-Linking Reagents</subject><subject>Genetic Variation</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Membrane Proteins - chemistry</subject><subject>Microfilament Proteins - chemistry</subject><subject>Molecular Sequence Data</subject><subject>Muscle, Skeletal - metabolism</subject><subject>Mutagenesis, Site-Directed</subject><subject>Peptide Fragments - chemistry</subject><subject>Phosphoproteins</subject><subject>Protein Structure, Secondary</subject><subject>Rabbits</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Homology, Amino Acid</subject><subject>Thymosin - chemistry</subject><subject>Thymosin - isolation & purification</subject><subject>Thymosin - metabolism</subject><issn>0261-4189</issn><issn>1460-2075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkctOwzAQRS0EKqXwCUgWC3YJ4zzsGIkFVKWAitiUteUkdusqjxK70Pw9Ca0qWLIZj3Tmzlj3InRFwCcAwc3KJxEFLwAW-4Rz6rsUIIzB3x6h4QEdoyEElHgRSfgpOrN2BQBxwsgADRJKGOHxEE3nS4Vl5kyFU1Plplpga5zCtcZu2Za17YFyEke4lOu1ynHa4nLjpDN1JQssu9JaY8_RiZaFVRf7d4TeHyfz8ZM3e5s-j-9nXhYDA09RHmgAFuSSMkq01DxKEwU0kVITCjqP0pQxnvA4i3XWUcUZCblOYplT4OEI3e32rjdpqfJMVa6RhVg3ppRNK2ppxF9SmaVY1J8iijgPo05_vdc39cdGWSdKYzNVFLJS9cYKwjqDKafd4O1uMGtqaxulDzcIiD4FsRK91aK3WvQpiH0KYtuJL3__8iDd297x-x3_MoVq_7FZTF4fXn768BvGyJk9</recordid><startdate>19960115</startdate><enddate>19960115</enddate><creator>Van Troys, M.</creator><creator>Dewitte, D.</creator><creator>Goethals, M.</creator><creator>Carlier, M. F.</creator><creator>Vandekerckhove, J.</creator><creator>Ampe, C.</creator><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>7TM</scope><scope>5PM</scope></search><sort><creationdate>19960115</creationdate><title>The actin binding site of thymosin beta 4 mapped by mutational analysis</title><author>Van Troys, M. ; Dewitte, D. ; Goethals, M. ; Carlier, M. F. ; Vandekerckhove, J. ; Ampe, C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5070-e692f0072da6761faf94b8e068aaf160fd4bb779895c5fcf94e97139f85ad6093</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Actins - chemistry</topic><topic>Actins - isolation & purification</topic><topic>Actins - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Binding, Competitive</topic><topic>Blood Proteins - chemistry</topic><topic>Calcium-Binding Proteins - chemistry</topic><topic>Carrier Proteins - chemistry</topic><topic>Chickens</topic><topic>Circular Dichroism</topic><topic>Conserved Sequence</topic><topic>Cross-Linking Reagents</topic><topic>Genetic Variation</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Membrane Proteins - chemistry</topic><topic>Microfilament Proteins - chemistry</topic><topic>Molecular Sequence Data</topic><topic>Muscle, Skeletal - metabolism</topic><topic>Mutagenesis, Site-Directed</topic><topic>Peptide Fragments - chemistry</topic><topic>Phosphoproteins</topic><topic>Protein Structure, Secondary</topic><topic>Rabbits</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Homology, Amino Acid</topic><topic>Thymosin - chemistry</topic><topic>Thymosin - isolation & purification</topic><topic>Thymosin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Van Troys, M.</creatorcontrib><creatorcontrib>Dewitte, D.</creatorcontrib><creatorcontrib>Goethals, M.</creatorcontrib><creatorcontrib>Carlier, M. 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F.</au><au>Vandekerckhove, J.</au><au>Ampe, C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The actin binding site of thymosin beta 4 mapped by mutational analysis</atitle><jtitle>The EMBO journal</jtitle><addtitle>EMBO J</addtitle><date>1996-01-15</date><risdate>1996</risdate><volume>15</volume><issue>2</issue><spage>201</spage><epage>210</epage><pages>201-210</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><abstract>We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross‐linking, complex formation in native gels and actin‐sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N‐terminal part needs to adopt an alpha‐helix for actin binding and interacts through a patch of hydrophobic residues (6M‐I‐F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin.</abstract><cop>England</cop><pmid>8617195</pmid><doi>10.1002/j.1460-2075.1996.tb00350.x</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0261-4189 |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Actins - chemistry Actins - isolation & purification Actins - metabolism Amino Acid Sequence Animals Binding Sites Binding, Competitive Blood Proteins - chemistry Calcium-Binding Proteins - chemistry Carrier Proteins - chemistry Chickens Circular Dichroism Conserved Sequence Cross-Linking Reagents Genetic Variation Humans Kinetics Membrane Proteins - chemistry Microfilament Proteins - chemistry Molecular Sequence Data Muscle, Skeletal - metabolism Mutagenesis, Site-Directed Peptide Fragments - chemistry Phosphoproteins Protein Structure, Secondary Rabbits Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sequence Homology, Amino Acid Thymosin - chemistry Thymosin - isolation & purification Thymosin - metabolism |
| title | The actin binding site of thymosin beta 4 mapped by mutational analysis |
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