Functional mapping of protein-protein interactions in an enzyme complex by directed evolution

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• The shikimate pathway is essential for producing aromatic amino acids Tyr and Phe.
• Mycobacterium tuberculosis chorismate mutase (MtCM) exhibits low intrinsic activity.
• MtCM activity dramatically increases (>100-fold) upon complex formation with 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS).

Purpose of the Study:

• To investigate the role of C-terminal residues (84-90) of MtCM in its interaction and activation by MtDS.
• To understand the evolutionary constraints and functional significance of specific amino acid residues at the MtCM-MtDS interface.
• To identify potential drug targets by mapping critical protein-protein interaction sites.

Main Methods:

• Employing evolutionary strategies with in vivo selection to probe MtCM C-terminal residue substitutions.
• Analyzing sequence patterns in active library members and comparing them to conserved residues in natural chorismate mutases.
• Purifying MtCM variants using a novel plasmid-based T7 RNA polymerase gene expression system.
• Assessing the physical interaction, activatability, and feedback regulation of MtCM variants with MtDS.

Main Results:

• Specific C-terminal residues (84-90) of MtCM are critical for functional interaction with MtDS.
• An invariant Arg-Gly dyad at positions 85-86 is intolerant to mutation.
• Leu88 and Gly89 prefer small, hydrophobic residues for optimal MtCM-MtDS complex formation.
• Residues 84-86 determine MtCM integrity in the absence of MtDS, while more C-terminal residues are involved in MtDS-mediated activation.
• Diminished MtCM-MtDS interaction correlates with reduced activatability and feedback inhibition.

Conclusions:

• Evolutionary strategies effectively map critical protein-protein interaction sites.
• The C-terminus of MtCM plays a dual role in maintaining enzyme integrity and mediating activation by MtDS.
• Understanding these interactions provides a basis for designing drugs that disrupt MtCM-MtDS complex formation, potentially inhibiting essential pathways in Mycobacterium tuberculosis.