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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
Karolinska University Hospital.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan.ORCID-id: 0000-0002-7001-9415
Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
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2010 (Engelska)Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, nr 4, s. 680-692Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Cold Spring Harbor Laboratory Press , 2010. Vol. 19, nr 4, s. 680-692
Nyckelord [en]
DNA-binding protein, stability, efflux gene regulator, multidrug resistance, MarR family, Biacore, analytical ultracentrifugation, circular dichroism, fluorescence, real-time PCR
Nationell ämneskategori
Teknik och teknologier
Identifikatorer
URN: urn:nbn:se:liu:diva-54849DOI: 10.1002/pro.343ISI: 000276274900006OAI: oai:DiVA.org:liu-54849DiVA, id: diva2:310867
Tillgänglig från: 2010-04-16 Skapad: 2010-04-16 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
Ingår i avhandling
1. Protein Structure and Interaction in Health and Disease
Öppna denna publikation i ny flik eller fönster >>Protein Structure and Interaction in Health and Disease
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2011. s. 67
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1394
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:liu:diva-70837 (URN)978-91-7393-077-2 (ISBN)
Disputation
2011-10-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 14:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2011-09-20 Skapad: 2011-09-20 Senast uppdaterad: 2019-12-19Bibliografiskt granskad

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

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Andrésen, CeciliaAili, DanielJarl, AnngelicaLiedberg, BoMårtensson, Lars-GöranSunnerhagen, Maria
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Molekylär BioteknikTekniska högskolanSensorvetenskap och MolekylfysikInstitutionen för fysik, kemi och biologiMolekylär genetikBiokemi
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