E. coli Multiple Antibiotic Resistance Regulator Protein

Erick Ditmars '18 and Kay Burrows '18


I. Introduction

The Escherichia coli Multiple antibiotic resistance regulator protein (MarR) is the hallmark member of the MarR family, responsible for regulating genes involved in antibiotic resistance. The MarR family of proteins is found in both prokaryotes and archaea and have been shown to play a significant role in the development of antibiotic resistant bacterial strains that pose a severe threat to global healthcare. E. coli's MarR protein acts as a negative repressor of the MarRAB system

When this system is activated, its primary active protein, MarA, acts as a transcriptional factor, upregulating 60 resistance genes. These genes typically encode for proteins involved in the construction of multi-drug efflux pumps, which allowing the organism to resist not only antibiotics, but also other molecules such as oxidative agents or disinfectants.

In its native form, the MarR protein binds to the Mar operator upstream of the marA gene. When bound, the protein acts as a steric hinderance to RNA polymerase, blocking transcription. MarR also has a strong affinity to aromatic acid molecules that, when bound, inactivate the operator binding affinity of the MarR protein. This lifts the steric block, allowing for the transcription of MarA and the formation of the cell's antibiotic resistance phenotype. 


MarRAB Operon

Figure 1. The MarRAB operon by Duval et al.

II. General Structure

MarR is a comprised of two identical polypeptide chains with approximate overall dimensions of 50 X 55 X 45 Angstroms. Each subunit is made up of 6 and three beta pleated . The two subunits that form the dimer are held together by a linkage between the domain and the domain. In the N-terminal and C-terminal domains form a linker between the subunits of the protein, stabilizing the complex. The dimer is further stabilized in this region through hydrogen bonding between as well as interactions between . Another important linker between the two subunits comes from the interaction of which attract each other, holding the complex together.  

III. DNA binding



MarR Binding Sites

Figure 1. The MarR binding sites by Alekshun et al.

While each of the precise interactions have yet to be identified, many studies have concluded that amino acids 61-121 of MarR form a DNA binding domain using the binding motif. The motif binds to the phosphodiester backbone, stabalizing the complex while the motif binds within the major groove and is used for sequence recognition of two sites on MarO. At this point in time the exact method of protein-DNA interaction in this protein is unknown, but a mutation within the a4 helix eliminates the proteins DNA binding ability.

In addition, a "superrepressor" mutation of , between the a4 and a5 helices; as been shown to increase binding by upwards of 30 times. (G95 on one subunit could not be shown due to errors in data collection using the electron microscope). While these amino acids are known to play a role in binding, the specific binding motif is unknown. Currently, there are two competeing theroies on how MarR binds with DNA. The first model is predicated off of the winged-helix binding of the E2F-DP transcriptional factor heterodimer. In this model, MarR binds in two half-faces, one per each subunit. These half-faces bind on diffrent faces of the DNA double helix. Both of these half-faces bind with the major groove and play a role in sequence recognition. The second binding motif is reminiscent of DtxR binding. This second binding motif is similar to that of E2F-DP except that the half faces bind on the same face of the DNA helix.

IV. Salicylate binding

Sodium salicylate, a model inhibitor for MarR, is found to disrupt the protein’s activity in vitro and in cells, and therefore can be used to identify potential ligand binding domains in the protein. Some studies hypothesize that salicylate binds two sites on a single subunit of MarR, SAL-A and SAL-B, located on either side of the

In the proposed , the interaction is comprised of hydrogen bonds with Thr 72 and Arg 86, with the salicylate ring positioned over the hydrophobic chain of Pro 57. Similarly, in the proposed , the salicylate interacts through hydrogen bonds with Ala 70 and Arg 77 , with a hydrophobic Met 74 located under the salicylate ring. Due to their proximity to the DNA-binding helices, both sites are hypothesized to cause a conformational change in the DNA binding domains, disrupting binding activity and rendering the MarR molecule inactive.

However, a study by Duval et. al (2013) demonstrate that mutations in these regions did not result in a change in salicylate binding, suggesting these are not the physiological binding sites, but rather simply sites important for DNA binding. This study, through mutagenesis, suggests that were of greater importance to salicylate binding. These newly identified amino acids create a hypothesized ligand binding site between the DNA binding domain and the N-terminal and C-terminal dimerization domains.

VI. References

Alekshun MN, Levy SB, Mealy TR, Seaton BA, Head JF. The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3 A resolution. Nat Struct Biol. 2001 Aug;8(8):710-4.

Duval V, McMurry LM, Foster K, Head JF, Levy SB. A mutational analysis of the multiple antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA-binding domains. J Bacteriol. 2013;195:3341–3351.253: 1001-1007.

Wilkinson, S. P. and Grove, A. ( 2006 ). Ligand-responsive transcriptional regulation by members of the MarR family of winged helix proteins. Curr Issues Mol Biol 8, 51–62.

Martin R.G., Rosner J.L (1995) Binding of purified multiple antibiotic resistance repressor protein (MarR) to mar operator sequences. Proc. Natl. Acad. Sci. USA 92, 5456–5460.

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