Membrane-embedded CdaA is required for efficient synthesis of second messenger cyclic di-AMP

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Cyclic di-adenylate monophosphate (cyclic di-AMP) is a crucial second messenger in microorganisms.
• It regulates bacterial cell volume, turgor, and innate immune responses.

Purpose of the Study:

• To compare the activity of full-length membrane-embedded CdaA with its soluble catalytic domain (CdaA-DAC).
• To elucidate the structure and functional characteristics of the membrane-bound CdaA enzyme.

Main Methods:

• Purification and study of CdaA from L. lactis in detergent-solubilized state, lipid nanodiscs, and vesicles.
• Cryo-electron microscopy (Cryo-EM) and in-silico structure prediction.
• Enzyme activity assays under varying pH, salt, and lipid conditions.

Main Results:

• Membrane-bound CdaA is tetrameric and exhibits over 100-fold higher activity than soluble CdaA-DAC.
• CdaA activity is pH-dependent but not strongly influenced by salt or lipid content.
• Structural analysis revealed a head-to-head interaction of DAC dimers forming the active cyclase, with dynamic flexibility observed.

Conclusions:

• Full-length membrane-bound CdaA is a highly active enzyme with a unique tetrameric structure.
• Understanding CdaA's structure and membrane association provides insights into cyclic di-AMP synthesis regulation.
• This work offers the first comprehensive characterization of a full-length cyclic di-AMP cyclase.