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Updated: May 6, 2026
Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Published on: December 30, 2016
Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a
Nayoung Kwon1, SooHo Rho1, Sung Chul Ha2
1School of Systems Biomedical Science and Integrative Institute of Basic Sciences, Soongsil University, Seoul 06978, Republic of Korea.
Abstract:
Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.
Insights
Clostridioides difficile MarR CD0473 protein
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Clostridioides difficile infection (CDI) is a growing public health concern due to increasing antibiotic resistance.
- MarR (Multiple antibiotic resistance Regulator) proteins regulate bacterial stress responses, but their function in C. difficile is largely uncharacterized.
- Understanding C. difficile MarR proteins is crucial for developing new therapeutic strategies against CDI.
Purpose of the Study:
- To elucidate the function and regulatory mechanism of the C. difficile MarR protein, CD0473.
- To investigate the structural basis for CD0473's interaction with DNA and its potential role in redox regulation.
Main Methods:
- X-ray crystallography was used to determine the structure of Hg-bound C. difficile MarR CD0473.
- Electrophoretic mobility shift assays (EMSAs) were performed to assess protein-DNA binding.
- Comparative analysis of protein structure and DNA-binding data under different redox conditions.
Main Results:
- The X-ray crystal structure revealed two flexible conformations of Hg-bound cysteines in CD0473, suggesting potential disulfide bridge formation.
- Two pseudo-palindromic DNA binding sites were identified in the intergenic regions near CD0473.
- CD0473 exhibited stronger DNA binding under oxidizing conditions, supporting a redox-regulated function.
Conclusions:
- C. difficile MarR CD0473 likely functions as a redox-regulated transcriptional switch.
- Disulfide bond formation under oxidizing conditions may modulate CD0473's DNA-binding activity.
- This study provides structural and functional insights into a novel regulatory mechanism in C. difficile.
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