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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Karolinska University Hospital.
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7001-9415
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
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2010 (English)In: Protein Science, ISSN 0961-8368, Vol. 19, no 4, 680-692 p.Article in journal (Refereed) Published
Abstract [en]

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

Place, publisher, year, edition, pages
Cold Spring Harbor Laboratory Press , 2010. Vol. 19, no 4, 680-692 p.
Keyword [en]
DNA-binding protein, stability, efflux gene regulator, multidrug resistance, MarR family, Biacore, analytical ultracentrifugation, circular dichroism, fluorescence, real-time PCR
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-54849DOI: 10.1002/pro.343ISI: 000276274900006OAI: diva2:310867
Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2014-10-08Bibliographically approved
In thesis
1. Protein Structure and Interaction in Health and Disease
Open this publication in new window or tab >>Protein Structure and Interaction in Health and Disease
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on protein structure, dynamics and interaction and their relation to human disease. In particular, the biophysical and structural properties of both well-ordered and partially disordered proteins are studied using a range of biophysical techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. Pseudomonas aeruginosa is a human pathogen due to its multidrug resistance (MDR) caused by overexpression of efflux pump systems. This thesis describes how MDR mutations within the MexR repressor of the MexAB-OprM system reduce the DNA affinity by altering its stability with maintained structure. The oncogenic protein c-Myc is involved in many essential biological functions such as cell proliferation, differentiation and apoptosis and is also highly associated with several forms of human cancers, and where the N-terminal domain is regulated by a plethora of protein interactions. In this thesis the intrinsically disordered N-terminal part of c-Myc and its interactions with the proteins Bin1 and TBP are described. Myc binds Bin1 with maintained disorder in a multivalent manner, which may explain why the onco-protein can interact with such a wide range of binding partners. A similarly dynamic interaction is observed for Myc with the TATA-binding protein (TBP). The essential human multidomain glutaredoxin Grx3 is associated with several biological functions such as redox signaling, proliferation and signal transduction. We have solved the structure and analyzed the dynamic properties in the ps-ns and ms time scale for the two N-terminal domains, providing a platform for further analysis of the Grx3 protein and its interactions. Taken together, this thesis emphasizes the importance of joint structural, biophysical and dynamic studies to better understand protein function in health and disease.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 67 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1394
National Category
Natural Sciences
urn:nbn:se:liu:diva-70837 (URN)978-91-7393-077-2 (ISBN)
Public defence
2011-10-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 14:00 (Swedish)
Available from: 2011-09-20 Created: 2011-09-20 Last updated: 2011-09-20Bibliographically approved

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Andrésen, CeciliaAili, DanielJarl, AnngelicaLiedberg, BoMårtensson, Lars-GöranSunnerhagen, Maria
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