The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>

ABSTRACT Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA o...

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Autores principales:	Ivan Birukou, Nam K. Tonthat, Susan M. Seo, Bryan D. Schindler, Glenn W. Kaatz, Richard G. Brennan
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/07f73ecbe7c84e91b1c2658cca5f9fe4
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spelling	oai:doaj.org-article:07f73ecbe7c84e91b1c2658cca5f9fe42021-11-15T15:42:47ZThe Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>10.1128/mBio.00528-132150-7511https://doaj.org/article/07f73ecbe7c84e91b1c2658cca5f9fe42013-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00528-13https://doaj.org/toc/2150-7511ABSTRACT Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus. IMPORTANCE Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.Ivan BirukouNam K. TonthatSusan M. SeoBryan D. SchindlerGlenn W. KaatzRichard G. BrennanAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 5 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Ivan Birukou Nam K. Tonthat Susan M. Seo Bryan D. Schindler Glenn W. Kaatz Richard G. Brennan The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
description	ABSTRACT Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus. IMPORTANCE Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.
format	article
author	Ivan Birukou Nam K. Tonthat Susan M. Seo Bryan D. Schindler Glenn W. Kaatz Richard G. Brennan
author_facet	Ivan Birukou Nam K. Tonthat Susan M. Seo Bryan D. Schindler Glenn W. Kaatz Richard G. Brennan
author_sort	Ivan Birukou
title	The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
title_short	The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
title_full	The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
title_fullStr	The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
title_full_unstemmed	The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>
title_sort	molecular mechanisms of allosteric mutations impairing mepr repressor function in multidrug-resistant strains of <named-content content-type="genus-species">staphylococcus aureus</named-content>
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/07f73ecbe7c84e91b1c2658cca5f9fe4
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The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of <named-content content-type="genus-species">Staphylococcus aureus</named-content>

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