Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization
The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of pote...
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| Veröffentlicht in: | Journal of biological inorganic chemistry 2016-10, Vol.21 (7), p.875-886 |
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| creator | Draganova, Elizabeth B. Adrian, Seth A. Lukat-Rodgers, Gudrun S. Keutcha, Cyrianne S. Schmitt, Michael P. Rodgers, Kenton R. Dixon, Dabney W. |
| description | The heme-binding protein HmuT is part of the
Corynebacterium diphtheriae
heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on
π
donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO. |
| doi_str_mv | 10.1007/s00775-016-1386-3 |
| format | Article |
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Corynebacterium diphtheriae
heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on
π
donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO.</description><identifier>ISSN: 0949-8257</identifier><identifier>EISSN: 1432-1327</identifier><identifier>DOI: 10.1007/s00775-016-1386-3</identifier><identifier>PMID: 27561288</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>ABC transporter ; Absorbance ; Arginine ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biochemistry ; Biomedical and Life Sciences ; Conserved Sequence ; Heme ; Hydrogen Bonding ; Life Sciences ; Ligands ; Lipoproteins - chemistry ; Lipoproteins - genetics ; Lipoproteins - metabolism ; Mass spectroscopy ; Methionine ; Microbiology ; Models, Molecular ; Mutagenesis, Site-Directed ; Mutation ; Nucleotide sequence ; Original Paper ; Phenols ; Protein folding ; Protein Stability ; Protein Structure, Secondary ; Protein Unfolding ; Proteins ; Sequence Alignment ; Temperature ; Thermal stability ; Tyrosine</subject><ispartof>Journal of biological inorganic chemistry, 2016-10, Vol.21 (7), p.875-886</ispartof><rights>SBIC 2016</rights><rights>Copyright Springer Science & Business Media 2016</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-b67d6c5eff8df1a5dc64a7fd70260a287f68cc210b7b2c4465fddd95d4937a573</citedby><cites>FETCH-LOGICAL-c372t-b67d6c5eff8df1a5dc64a7fd70260a287f68cc210b7b2c4465fddd95d4937a573</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00775-016-1386-3$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00775-016-1386-3$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27561288$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Draganova, Elizabeth B.</creatorcontrib><creatorcontrib>Adrian, Seth A.</creatorcontrib><creatorcontrib>Lukat-Rodgers, Gudrun S.</creatorcontrib><creatorcontrib>Keutcha, Cyrianne S.</creatorcontrib><creatorcontrib>Schmitt, Michael P.</creatorcontrib><creatorcontrib>Rodgers, Kenton R.</creatorcontrib><creatorcontrib>Dixon, Dabney W.</creatorcontrib><title>Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization</title><title>Journal of biological inorganic chemistry</title><addtitle>J Biol Inorg Chem</addtitle><addtitle>J Biol Inorg Chem</addtitle><description>The heme-binding protein HmuT is part of the
Corynebacterium diphtheriae
heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on
π
donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO.</description><subject>ABC transporter</subject><subject>Absorbance</subject><subject>Arginine</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Conserved Sequence</subject><subject>Heme</subject><subject>Hydrogen Bonding</subject><subject>Life Sciences</subject><subject>Ligands</subject><subject>Lipoproteins - chemistry</subject><subject>Lipoproteins - genetics</subject><subject>Lipoproteins - metabolism</subject><subject>Mass spectroscopy</subject><subject>Methionine</subject><subject>Microbiology</subject><subject>Models, Molecular</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>Nucleotide sequence</subject><subject>Original Paper</subject><subject>Phenols</subject><subject>Protein folding</subject><subject>Protein Stability</subject><subject>Protein Structure, Secondary</subject><subject>Protein Unfolding</subject><subject>Proteins</subject><subject>Sequence Alignment</subject><subject>Temperature</subject><subject>Thermal stability</subject><subject>Tyrosine</subject><issn>0949-8257</issn><issn>1432-1327</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1PGzEQhq2qCNKUH9BLZakXLgv-WH-ktyqiBQmJS3q2vPaYGHbXqb2LFH49RoGqqsTFY88883o0L0JfKDmnhKiLUg8lGkJlQ7mWDf-AFrTlrL6Y-ogWZNWuGs2EOkGfSrknhHBBxTE6YUpIyrReoLhOeT9CZ90EOc4D9nG3nbb1bgFfDfPme82UAm6K4x2uBZxTDwWngF0aC-RH8DhDiX6u2TjiLQyAd8k9wITLZLvYxyc7xTR-RkfB9gVOX-MS_f55uVlfNTe3v67XP24axxWbmk4qL52AELQP1ArvZGtV8IowSSzTKkjtHKOkUx1zbStF8N6vhG9XXFmh-BKdHXR3Of2pQ01miMVB39sR0lwM1YwT3nJFK_rtP_Q-zXms01VKEy1VS2Wl6IFyOZWSIZhdjoPNe0OJefHBHHww1Qfz4oPhtefrq_LcDeD_drwtvgLsAJRaGu8g__P1u6rP-F6UXA</recordid><startdate>20161001</startdate><enddate>20161001</enddate><creator>Draganova, Elizabeth B.</creator><creator>Adrian, Seth A.</creator><creator>Lukat-Rodgers, Gudrun S.</creator><creator>Keutcha, Cyrianne S.</creator><creator>Schmitt, Michael P.</creator><creator>Rodgers, Kenton R.</creator><creator>Dixon, Dabney W.</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature B.V</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>7X8</scope></search><sort><creationdate>20161001</creationdate><title>Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization</title><author>Draganova, Elizabeth B. ; Adrian, Seth A. ; Lukat-Rodgers, Gudrun S. ; Keutcha, Cyrianne S. ; Schmitt, Michael P. ; Rodgers, Kenton R. ; Dixon, Dabney W.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-b67d6c5eff8df1a5dc64a7fd70260a287f68cc210b7b2c4465fddd95d4937a573</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>ABC transporter</topic><topic>Absorbance</topic><topic>Arginine</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Conserved Sequence</topic><topic>Heme</topic><topic>Hydrogen Bonding</topic><topic>Life Sciences</topic><topic>Ligands</topic><topic>Lipoproteins - chemistry</topic><topic>Lipoproteins - genetics</topic><topic>Lipoproteins - metabolism</topic><topic>Mass spectroscopy</topic><topic>Methionine</topic><topic>Microbiology</topic><topic>Models, Molecular</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>Nucleotide sequence</topic><topic>Original Paper</topic><topic>Phenols</topic><topic>Protein folding</topic><topic>Protein Stability</topic><topic>Protein Structure, Secondary</topic><topic>Protein Unfolding</topic><topic>Proteins</topic><topic>Sequence Alignment</topic><topic>Temperature</topic><topic>Thermal stability</topic><topic>Tyrosine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Draganova, Elizabeth B.</creatorcontrib><creatorcontrib>Adrian, Seth A.</creatorcontrib><creatorcontrib>Lukat-Rodgers, Gudrun S.</creatorcontrib><creatorcontrib>Keutcha, Cyrianne S.</creatorcontrib><creatorcontrib>Schmitt, Michael P.</creatorcontrib><creatorcontrib>Rodgers, Kenton R.</creatorcontrib><creatorcontrib>Dixon, Dabney 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>MEDLINE - Academic</collection><jtitle>Journal of biological inorganic chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Draganova, Elizabeth B.</au><au>Adrian, Seth A.</au><au>Lukat-Rodgers, Gudrun S.</au><au>Keutcha, Cyrianne S.</au><au>Schmitt, Michael P.</au><au>Rodgers, Kenton R.</au><au>Dixon, Dabney W.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization</atitle><jtitle>Journal of biological inorganic chemistry</jtitle><stitle>J Biol Inorg Chem</stitle><addtitle>J Biol Inorg Chem</addtitle><date>2016-10-01</date><risdate>2016</risdate><volume>21</volume><issue>7</issue><spage>875</spage><epage>886</epage><pages>875-886</pages><issn>0949-8257</issn><eissn>1432-1327</eissn><abstract>The heme-binding protein HmuT is part of the
Corynebacterium diphtheriae
heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on
π
donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>27561288</pmid><doi>10.1007/s00775-016-1386-3</doi><tpages>12</tpages></addata></record> |
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| subjects | ABC transporter Absorbance Arginine Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Biochemistry Biomedical and Life Sciences Conserved Sequence Heme Hydrogen Bonding Life Sciences Ligands Lipoproteins - chemistry Lipoproteins - genetics Lipoproteins - metabolism Mass spectroscopy Methionine Microbiology Models, Molecular Mutagenesis, Site-Directed Mutation Nucleotide sequence Original Paper Phenols Protein folding Protein Stability Protein Structure, Secondary Protein Unfolding Proteins Sequence Alignment Temperature Thermal stability Tyrosine |
| title | Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization |
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