Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA
Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more close...
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Veröffentlicht in: | Biochemical journal 2008-07, Vol.413 (1), p.93-101 |
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description | Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIalpha 92-260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIalpha 92-260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIalpha and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied. |
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Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIalpha 92-260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIalpha 92-260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIalpha and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.</description><identifier>ISSN: 0264-6021</identifier><identifier>EISSN: 1470-8728</identifier><identifier>DOI: 10.1042/BJ20071665</identifier><identifier>PMID: 18373497</identifier><language>eng</language><publisher>England: Portland Press</publisher><subject>Binding, Competitive - drug effects ; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism ; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors ; Cyclic AMP-Dependent Protein Kinases - chemistry ; Enzyme Inhibitors - metabolism ; Magnesium - pharmacology ; Models, Molecular ; Nucleotides - metabolism ; Peptides - metabolism ; Protein Binding - drug effects ; Protein Conformation ; Protein Subunits ; Surface Plasmon Resonance</subject><ispartof>Biochemical journal, 2008-07, Vol.413 (1), p.93-101</ispartof><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-751cacbffd55a200944bf5d82dedfb9e8a8ea9aa8f3bebf80e7865784f1fd0b63</citedby><cites>FETCH-LOGICAL-c386t-751cacbffd55a200944bf5d82dedfb9e8a8ea9aa8f3bebf80e7865784f1fd0b63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18373497$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-00478937$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Zimmermann, Bastian</creatorcontrib><creatorcontrib>Schweinsberg, Sonja</creatorcontrib><creatorcontrib>Drewianka, Stephan</creatorcontrib><creatorcontrib>Herberg, Friedrich W</creatorcontrib><title>Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA</title><title>Biochemical journal</title><addtitle>Biochem J</addtitle><description>Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIalpha 92-260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIalpha 92-260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIalpha and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.</description><subject>Binding, Competitive - drug effects</subject><subject>Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism</subject><subject>Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors</subject><subject>Cyclic AMP-Dependent Protein Kinases - chemistry</subject><subject>Enzyme Inhibitors - metabolism</subject><subject>Magnesium - pharmacology</subject><subject>Models, Molecular</subject><subject>Nucleotides - metabolism</subject><subject>Peptides - metabolism</subject><subject>Protein Binding - drug effects</subject><subject>Protein Conformation</subject><subject>Protein Subunits</subject><subject>Surface Plasmon Resonance</subject><issn>0264-6021</issn><issn>1470-8728</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkdFKwzAUhoMobk5vfADJlaBQPWnSJr2cYzp1oIhel6RN1kjbzCYV9vZ2bOilVwcOHz_n_B9C5wRuCLD49u4pBuAkTZMDNCaMQyR4LA7RGOKURSnEZIROvP8EIAwYHKMREZRTlvExepsbo4uAncGNDrLG1rUeuxZXdlVF0hjb2rDByralbVdbbO11XzrfKx86GTS2bWWVDa7zODj8-jw9RUdG1l6f7ecEfdzP32eLaPny8DibLqOCijREPCGFLJQxZZLI4YGMMWWSUsSlLo3KtJBCy0xKYajSygjQXKQJF8wQU4JK6QRd7XIrWefrzjay2-RO2nwxXebbHQDjIqP8mwzs5Y5dd-6r1z7kjfWFrmvZatf7fGgvAZLRf8EYBI3F0N8EXe_AonPed9r8nkAg32rJ_7QM8MU-tVeNLv_QvQf6A7Nahz0</recordid><startdate>20080701</startdate><enddate>20080701</enddate><creator>Zimmermann, Bastian</creator><creator>Schweinsberg, Sonja</creator><creator>Drewianka, Stephan</creator><creator>Herberg, Friedrich W</creator><general>Portland Press</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>7TM</scope><scope>7X8</scope><scope>1XC</scope><scope>VOOES</scope></search><sort><creationdate>20080701</creationdate><title>Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA</title><author>Zimmermann, Bastian ; Schweinsberg, Sonja ; Drewianka, Stephan ; Herberg, Friedrich W</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-751cacbffd55a200944bf5d82dedfb9e8a8ea9aa8f3bebf80e7865784f1fd0b63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Binding, Competitive - drug effects</topic><topic>Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism</topic><topic>Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors</topic><topic>Cyclic AMP-Dependent Protein Kinases - chemistry</topic><topic>Enzyme Inhibitors - metabolism</topic><topic>Magnesium - pharmacology</topic><topic>Models, Molecular</topic><topic>Nucleotides - metabolism</topic><topic>Peptides - metabolism</topic><topic>Protein Binding - drug effects</topic><topic>Protein Conformation</topic><topic>Protein Subunits</topic><topic>Surface Plasmon Resonance</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zimmermann, Bastian</creatorcontrib><creatorcontrib>Schweinsberg, Sonja</creatorcontrib><creatorcontrib>Drewianka, Stephan</creatorcontrib><creatorcontrib>Herberg, Friedrich W</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><collection>Hyper Article en Ligne (HAL) (Open Access)</collection><jtitle>Biochemical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zimmermann, Bastian</au><au>Schweinsberg, Sonja</au><au>Drewianka, Stephan</au><au>Herberg, Friedrich W</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA</atitle><jtitle>Biochemical journal</jtitle><addtitle>Biochem J</addtitle><date>2008-07-01</date><risdate>2008</risdate><volume>413</volume><issue>1</issue><spage>93</spage><epage>101</epage><pages>93-101</pages><issn>0264-6021</issn><eissn>1470-8728</eissn><abstract>Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIalpha 92-260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIalpha 92-260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIalpha and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.</abstract><cop>England</cop><pub>Portland Press</pub><pmid>18373497</pmid><doi>10.1042/BJ20071665</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Binding, Competitive - drug effects Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors Cyclic AMP-Dependent Protein Kinases - chemistry Enzyme Inhibitors - metabolism Magnesium - pharmacology Models, Molecular Nucleotides - metabolism Peptides - metabolism Protein Binding - drug effects Protein Conformation Protein Subunits Surface Plasmon Resonance |
title | Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA |
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