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Population shift disengages DNA binding in a multidrug resistance MexR mutant
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. School of Molecular Bioscience, The University of Sydney, New South Wales, Australia.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The homodimeric MexR protein, featuring a MarR helical dimer fold with two DNA-binding wHTH domains, functions as a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa. Mutations in MexR lead to multidrug resistance (MDR) by impairing MexR-directed repression of the efflux operon. In this study, we present the crystal structure of MexR mutant R21W at a resolution of 2.19 Å, together with an evaluation of MexR-wt and -R21W protein dynamics using molecular dynamics simulations. While the crystal structure MexR-R21W is closely similar to that of MexR-wt, our molecular simulations show that the mutant protein only accesses a subset of the conformational space available to the wt protein. Specifically, the R21W mutation results in the coupling of distinct allosteric contact networks that are independent in MexR-wt, effectively leading to reduced independent mobility of wHTH domains relative to the dimerization domain in the mutated protein. Experimental small-angle X-ray scattering in solution independently support a larger structural envelope than the crystal structure for both proteins, and more so for MexR-wt than for MexR-R21W. Taken together, our results suggest that the MexR-R21W mutation disengages DNA binding through population shift, and that this mimics the derepression effected by small-molecule binding to MarR proteins that confers antibiotics resistance. While this view contrasts the previously proposed structure-based cascade of rigid-body rotations that would lead to derepression in the MarR family, our population shift model is in full agreement with previously published data and may well be present also for other MarR proteins.

National Category
Chemical Sciences Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-122465OAI: oai:DiVA.org:liu-122465DiVA: diva2:866604
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-03Bibliographically approved
In thesis
1. Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
Open this publication in new window or tab >>Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.

The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in  nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.

In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.

Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.

Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 73 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1694
National Category
Chemical Sciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-122468 (URN)10.3384/diss.diva-122468 (DOI)978-91-7685-984-1 (print) (ISBN)
Public defence
2015-12-04, Planck, Fysikhuset, Campus Valla, Linköping, 09:30 (English)
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Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-17Bibliographically approved

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