Structure of Ribosome-Inactivating Protein from Mirabilis jalapa and Its L12-Stalk-Dependent Inhibition of...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Mirabilis antiviral protein (MAP) is a type I ribosome-inactivating protein (RIP).
• MAP uniquely inactivates both eukaryotic and E. coli ribosomes by cleaving 23S ribosomal RNA.
• The wild-type MAP structure remained uncharacterized.

Purpose of the Study:

• To determine the crystal structure of a recombinant MAP mutant.
• To elucidate the structural basis for MAP's ribosome inactivation mechanism.
• To identify key residues involved in MAP-RNA interaction.

Main Methods:

• Expression and purification of recombinant MAP mutants (MAP-EQRQ) in E. coli.
• X-ray crystallography to determine the crystal structure of MAP-EQRQ at 2.1 Å resolution.
• Quantitative RT-PCR to assess mutant protein activities and RNA binding.

Main Results:

• The crystal structure of MAP-EQRQ was determined at 2.1 Å resolution.
• Residue R171 was identified as a key active site residue for stable complex formation with adenine.
• MAP was shown to bind to the C-terminal domains of eukaryotic P2-stalk and E. coli L12-stalk.

Conclusions:

• The R171 residue is critical for MAP's interaction with the target adenine in ribosomes.
• MAP forms stable complexes by binding to specific stalk regions of both eukaryotic and bacterial ribosomes.
• This structural insight advances understanding of MAP's broad-spectrum ribosome inactivation mechanism.