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Crystal structure of the multiple antibiotic resistance regulator MarR from Clostridium difficile
1Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, People's Republic of China.
Abstract:
Regulators of multiple antibiotic resistance (MarRs) are key players against toxins in prokaryotes. MarR homologues have been identified in many bacterial and archaeal species which pose daunting antibiotic resistance issues that threaten public health. The continuous prevalence of Clostridium difficile infection (CDI) throughout the world is associated with the abuse of antibiotics, and antibiotic treatments of CDI have limited effect. In the genome of C. difficile strain 630, the marR gene (ID 4913953) encodes a MarR protein. Here, MarR from C. difficile (MarRC.difficile) was subcloned and crystallized for the first time. MarRC.difficile was successfully expressed in Escherichia coli in a soluble form and was purified to near-homogeneity (>95%) by a two-step purification protocol. The structure of MarRC.difficile has been solved at 2.3 Å resolution. The crystal belonged to the monoclinic space group P43212, with unit-cell parameters a = b = 66.569, c = 83.654 Å. The structure reported reveals MarRC.difficile to be a dimer, with each subunit consisting of six α-helices and three antiparallel β-hairpins. MarRC.difficile shows high structural similarity to the MarR proteins from E. coli and Staphylococcus aureus, indicating that MarRC.difficile might be a DNA-binding protein.
Insights
Regulators of multiple antibiotic resistance (MarRs) in Clostridium difficile were crystallized for the first time. This structural study of MarRC.difficile provides insights into antibiotic resistance mechanisms.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Regulators of multiple antibiotic resistance (MarRs) are crucial in prokaryotic defense against toxins.
- Antibiotic resistance poses a significant public health threat, with Clostridium difficile infections (CDI) being a major concern due to limited treatment efficacy.
- The marR gene in C. difficile strain 630 encodes a MarR protein implicated in antibiotic resistance.
Purpose of the Study:
- To characterize the structure of MarR from Clostridium difficile (MarRC.diff.) for the first time.
- To investigate the potential role of MarRC.diff. in antibiotic resistance mechanisms.
- To provide a structural basis for understanding MarR function in C. difficile.
Main Methods:
- Subcloning and expression of MarRC.diff. in Escherichia coli.
- Purification of MarRC.diff. to near-homogeneity using a two-step protocol.
- Crystallization and X-ray diffraction analysis to determine the 3D structure at 2.3 Å resolution.
Main Results:
- MarRC.diff. was successfully expressed, purified, and crystallized.
- The crystal structure of MarRC.diff. was solved, revealing a dimeric structure.
- Each subunit comprises six α-helices and three antiparallel β-hairpins, with high structural similarity to MarR proteins from E. coli and S. aureus.
Conclusions:
- The first crystal structure of MarRC.diff. has been determined.
- The structural similarity to other MarR proteins suggests MarRC.diff. is a DNA-binding protein.
- This finding contributes to understanding antibiotic resistance in C. difficile and may inform future therapeutic strategies.
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