Construction of a dimeric repressor: dissection of subunit interfaces in Lac repressor

Area of Science:

• Molecular Biology
• Protein Structure and Dynamics

Background:

• The lactose repressor protein (lac repressor) forms a tetramer through distinct subunit interfaces.
• A C-terminal heptad repeat of leucines is hypothesized to form an abbreviated coiled-coil motif crucial for dimer-dimer association.

Purpose of the Study:

• To investigate the role of the C-terminal coiled-coil motif in lac repressor tetramer formation.
• To strengthen the dimer-dimer interface by extending the leucine heptad repeat motif.

Main Methods:

• Site-specific insertion mutagenesis was used to add leucine heptad repeat units to the C-terminus.
• A point mutation (Y282D) was introduced to disrupt a different subunit interface, yielding a monomeric protein in the wild-type background.
• The assembly and functional properties of the modified repressors were analyzed.

Main Results:

• Extended coiled-coil mutants maintained wild-type operator and inducer binding affinities.
• Disruption of the other subunit interface still allowed dimer formation in the elongated mutants, indicating enhanced C-terminal association.
• These findings support the importance of the coiled-coil structure in tetramer assembly.

Conclusions:

• The C-terminal coiled-coil motif plays a significant role in the dimer-dimer association required for lac repressor tetramer formation.
• Strengthening this interface can facilitate dimer formation even when other interfaces are compromised.