Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)₃ template mediated strategy

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• Three-stranded coiled coils are common peptide structures.
• Achieving specific heterotrimer formation in coiled coils is challenging.

Purpose of the Study:

• To develop a method for creating pure heterotrimeric three-stranded coiled coils.
• To investigate the role of metal coordination and steric bulk in coiled coil assembly.
• To explore the potential for incorporating catalytic sites into these designed protein structures.

Main Methods:

• Utilizing a cysteine sulfur layer for metal coordination with lead(II) (Pb(II)).
• Engineering reduced steric bulk to create a water-binding cavity.
• Introducing specific cysteine substitutions at 'a' and 'd' sites to control heterotrimer formation.

Main Results:

• Pure Pb(II)A2B heterotrimers were formed by cysteine substitution at the 'a' site.
• Pure Pb(II)C2D or Pb(II)CD2 scaffolds were generated using 'd' sites.
• Altering metal or steric properties disrupted heterotrimer specificity.
• A zinc(II)-histidine catalytic center was successfully incorporated without affecting heterotrimer selectivity.

Conclusions:

• A novel strategy for constructing pure heterotrimeric coiled coils has been demonstrated.
• This method offers precise control over protein self-assembly.
• The designed scaffolds provide a platform for creating dissymmetric catalytic sites in de novo-designed proteins.