A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation

Autor:	Aimee Shen, Brian E. Eckenroth, Carolina Alves Feliciano, Sylvie Doublié, Oscar R. Diaz
Jazyk:	angličtina
Rok vydání:	2021
Předmět:	Cancer Research Polymers Regulator QH426-470 Crystallography X-Ray Biochemistry chemistry.chemical_compound Microbial Physiology Catalytic Domain Electrochemistry Salt Bridges Bacterial Physiology Enzyme Chemistry Materials Genetics (clinical) chemistry.chemical_classification Spores Bacterial Crystallography Molecular Structure Physics Monomers Condensed Matter Physics Chemistry Separation Processes Lytic cycle Macromolecules Muramic Acids Physical Sciences Crystal Structure Chromatography Gel Research Article Protein Binding Lactams Clostridium Difficile Lipoproteins Allosteric regulation Materials Science Biology Research and Analysis Methods Microbiology Catalysis Amidase Amidohydrolases Enzyme Regulation Allosteric Regulation Genetics Amidase activity Solid State Physics Bacterial Spores Molecular Biology Ecology Evolution Behavior and Systematics Bacteria Clostridioides difficile fungi Gut Bacteria Organisms Biology and Life Sciences Proteins Bacteriology Peptidoglycans Elution Polymer Chemistry Cortex (botany) Enzyme chemistry Enzymology Peptidoglycan
Zdroj:	PLoS Genetics PLoS Genetics, Vol 17, Iss 9, p e1009791 (2021)
ISSN:	1553-7404 1553-7390
Popis:	Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought. Author summary Spore germination is essential for many spore-forming pathogens to initiate infection. In order for spores to germinate, they must degrade a thick, protective layer of cell wall known as the cortex. The enzymes that digest this layer selectively recognize the spore-specific cell wall modification, muramic-∂-lactam (MAL). MAL is made in part through the activity of the CwlD amidase, which is found in all spore-forming bacteria. While Bacillus subtilis CwlD appears to have amidase activity on its own, Clostridioides difficile CwlD activity depends on its binding partner, the GerS lipoprotein. To understand why C. difficile CwlD requires GerS, we determined the X-ray crystal structure of the CwlD:GerS complex and discovered that GerS binds to a site distant from CwlD’s active site. We also found that GerS stabilizes CwlD binding to its co-factor, Zn2+, indicating that GerS allosterically activates CwlD amidase. Notably, regulation at the level of Zn2+ binding has not previously been described for bacterial amidases, and GerS is the first protein to be shown to allosterically activate an amidase. Since binding partners of bacterial amidases were only first discovered 15 years ago, our results suggest that diverse mechanisms remain to be discovered for these critical enzymes.
Databáze:	OpenAIRE
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