Characterization of Ejl, the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ejl amidase, encoded by the Ej-1 temperate phage, is a pneumococcal cell-wall lysin with a bimodular structure.
• The N-terminal module contains the catalytic region, while the C-terminal module mediates cell wall attachment via choline binding.

Purpose of the Study:

• To characterize the structural features and choline-binding interactions of Ejl amidase.
• To investigate how choline binding affects Ejl amidase structure and self-association.
• To compare Ejl amidase with the major pneumococcal amidase, LytA, to understand the impact of sequence divergence.

Main Methods:

• Circular dichroism (CD) and Infrared (IR) spectroscopies
• Differential scanning calorimetry (DSC)
• Analytical ultracentrifugation
• Fast Protein Liquid Chromatography (FPLC)

Main Results:

• Ejl amidase exhibits a predominantly beta-strand structure with limited alpha-helices in its catalytic module.
• Choline binding induces minor secondary structure changes but significantly enhances Ejl amidase self-association, favoring dimers and tetramers.
• Sequence differences (15% divergence) between Ejl and LytA amidases impact stability, domain organization, and choline-induced self-association, with Ejl showing a 10-fold lower ligand affinity.

Conclusions:

• Sequence variability in pneumococcal and bacteriophage cell wall amidases can significantly alter protein structure and cell wall attachment mechanisms.
• Ejl amidase's distinct structural and binding properties, compared to LytA, highlight the functional consequences of natural sequence divergence.