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Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding
The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydr...
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| Published in: | Antimicrobial agents and chemotherapy 2022-11, Vol.66 (11), p.e0067222-e0067222 |
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| container_end_page | e0067222 |
| container_issue | 11 |
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| container_title | Antimicrobial agents and chemotherapy |
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| creator | Yamasaki, Seiji Koga, Naoki Zwama, Martijn Sakurai, Keisuke Nakashima, Ryosuke Yamaguchi, Akihito Nishino, Kunihiko |
| description | The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery. |
| doi_str_mv | 10.1128/aac.00672-22 |
| format | article |
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Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.</description><identifier>ISSN: 0066-4804</identifier><identifier>EISSN: 1098-6596</identifier><identifier>DOI: 10.1128/aac.00672-22</identifier><identifier>PMID: 36300935</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Anti-Bacterial Agents - chemistry ; Bacterial Outer Membrane Proteins - metabolism ; Bacteriology ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Escherichia coli Proteins - metabolism ; Mechanisms of Resistance ; Membrane Transport Proteins - metabolism ; Multidrug Resistance-Associated Proteins - genetics ; Multidrug Resistance-Associated Proteins - metabolism ; Pseudomonas aeruginosa ; Tryptophan - pharmacology</subject><ispartof>Antimicrobial agents and chemotherapy, 2022-11, Vol.66 (11), p.e0067222-e0067222</ispartof><rights>Copyright © 2022 Yamasaki et al.</rights><rights>Copyright © 2022 Yamasaki et al. 2022 Yamasaki et al.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a484t-a8a20a04fc6272fd6b354019d260dc3cf21c5e69fe91f6cb13ac051f9a94ad213</citedby><cites>FETCH-LOGICAL-a484t-a8a20a04fc6272fd6b354019d260dc3cf21c5e69fe91f6cb13ac051f9a94ad213</cites><orcidid>0000-0001-9081-382X ; 0000-0003-3349-1769</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://journals.asm.org/doi/pdf/10.1128/aac.00672-22$$EPDF$$P50$$Gasm2$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://journals.asm.org/doi/full/10.1128/aac.00672-22$$EHTML$$P50$$Gasm2$$Hfree_for_read</linktohtml><link.rule.ids>230,315,733,786,790,891,3207,27957,27958,52786,52787,52788,53827,53829</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36300935$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yamasaki, Seiji</creatorcontrib><creatorcontrib>Koga, Naoki</creatorcontrib><creatorcontrib>Zwama, Martijn</creatorcontrib><creatorcontrib>Sakurai, Keisuke</creatorcontrib><creatorcontrib>Nakashima, Ryosuke</creatorcontrib><creatorcontrib>Yamaguchi, Akihito</creatorcontrib><creatorcontrib>Nishino, Kunihiko</creatorcontrib><title>Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding</title><title>Antimicrobial agents and chemotherapy</title><addtitle>Antimicrob Agents Chemother</addtitle><addtitle>Antimicrob Agents Chemother</addtitle><description>The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.</description><subject>Anti-Bacterial Agents - chemistry</subject><subject>Bacterial Outer Membrane Proteins - metabolism</subject><subject>Bacteriology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Mechanisms of Resistance</subject><subject>Membrane Transport Proteins - metabolism</subject><subject>Multidrug Resistance-Associated Proteins - genetics</subject><subject>Multidrug Resistance-Associated Proteins - metabolism</subject><subject>Pseudomonas aeruginosa</subject><subject>Tryptophan - pharmacology</subject><issn>0066-4804</issn><issn>1098-6596</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><recordid>eNp1kU1LxDAQhoMouq7ePEuOClaTtMk2F0HX9QMVBfUcpmmyzdIvk1b031tdFT14Gmbm4Z2ZdxDaoeSQUpYeAehDQsSERYytoBElMo0El2IVjYayiJKUJBtoM4QFGXIuyTraiEVMiIz5CF0_tNA5KPG0AA-6M96FzumAG4u7wuCZtWX_iu_7qsW35vUUn5mBqVxt8FVduMx1jcenrs5dPd9CaxbKYLa_4hg9nc8ep5fRzd3F1fTkJoIkTboIUmAESGK1YBNmc5HFPCFU5kyQXMfaMqq5EdIaSa3QGY1BE06tBJlAzmg8RsdL3bbPKpNrU3ceStV6V4F_Uw049bdTu0LNmxclhUhSLgaBvS8B3zz3JnSqckGbsoTaNH1Qw1qSUz4hH7MOlqj2TQje2J8xlKgP_9Xgv_r0XzE24PtLHELF1KLpfT048R-7-_uMH-Hv58TvRz2Onw</recordid><startdate>20221115</startdate><enddate>20221115</enddate><creator>Yamasaki, Seiji</creator><creator>Koga, Naoki</creator><creator>Zwama, Martijn</creator><creator>Sakurai, Keisuke</creator><creator>Nakashima, Ryosuke</creator><creator>Yamaguchi, Akihito</creator><creator>Nishino, Kunihiko</creator><general>American Society for Microbiology</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><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-9081-382X</orcidid><orcidid>https://orcid.org/0000-0003-3349-1769</orcidid></search><sort><creationdate>20221115</creationdate><title>Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding</title><author>Yamasaki, Seiji ; Koga, Naoki ; Zwama, Martijn ; Sakurai, Keisuke ; Nakashima, Ryosuke ; Yamaguchi, Akihito ; Nishino, Kunihiko</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a484t-a8a20a04fc6272fd6b354019d260dc3cf21c5e69fe91f6cb13ac051f9a94ad213</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Anti-Bacterial Agents - chemistry</topic><topic>Bacterial Outer Membrane Proteins - metabolism</topic><topic>Bacteriology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Mechanisms of Resistance</topic><topic>Membrane Transport Proteins - metabolism</topic><topic>Multidrug Resistance-Associated Proteins - genetics</topic><topic>Multidrug Resistance-Associated Proteins - metabolism</topic><topic>Pseudomonas aeruginosa</topic><topic>Tryptophan - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yamasaki, Seiji</creatorcontrib><creatorcontrib>Koga, Naoki</creatorcontrib><creatorcontrib>Zwama, Martijn</creatorcontrib><creatorcontrib>Sakurai, Keisuke</creatorcontrib><creatorcontrib>Nakashima, Ryosuke</creatorcontrib><creatorcontrib>Yamaguchi, Akihito</creatorcontrib><creatorcontrib>Nishino, Kunihiko</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><collection>PubMed Central (Full Participant titles)</collection><jtitle>Antimicrobial agents and chemotherapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yamasaki, Seiji</au><au>Koga, Naoki</au><au>Zwama, Martijn</au><au>Sakurai, Keisuke</au><au>Nakashima, Ryosuke</au><au>Yamaguchi, Akihito</au><au>Nishino, Kunihiko</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding</atitle><jtitle>Antimicrobial agents and chemotherapy</jtitle><stitle>Antimicrob Agents Chemother</stitle><addtitle>Antimicrob Agents Chemother</addtitle><date>2022-11-15</date><risdate>2022</risdate><volume>66</volume><issue>11</issue><spage>e0067222</spage><epage>e0067222</epage><pages>e0067222-e0067222</pages><issn>0066-4804</issn><eissn>1098-6596</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><notes>The authors declare no conflict of interest.</notes><notes>Present address: Keisuke Sakurai, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.</notes><abstract>The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>36300935</pmid><doi>10.1128/aac.00672-22</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0001-9081-382X</orcidid><orcidid>https://orcid.org/0000-0003-3349-1769</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0066-4804 |
| ispartof | Antimicrobial agents and chemotherapy, 2022-11, Vol.66 (11), p.e0067222-e0067222 |
| issn | 0066-4804 1098-6596 |
| language | eng |
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| source | American Society for Microbiology Journals; PubMed Central |
| subjects | Anti-Bacterial Agents - chemistry Bacterial Outer Membrane Proteins - metabolism Bacteriology Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - metabolism Mechanisms of Resistance Membrane Transport Proteins - metabolism Multidrug Resistance-Associated Proteins - genetics Multidrug Resistance-Associated Proteins - metabolism Pseudomonas aeruginosa Tryptophan - pharmacology |
| title | Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding |
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