Heteromeric assembled polypeptidic artificial hydrolases with a six-helical bundle scaffold

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Mimicry

Background:

• Enzyme efficiency relies on cooperative functional groups within a catalytic site.
• Mimicking natural enzymes requires a precisely designed 3D scaffold for functional group cooperation.
• The HIV-1 fusion process involves a six-helical bundle (6HB) formed by gp41 N- and C-terminal regions.

Purpose of the Study:

• To utilize the 6HB structure as a molecular model for designing a novel artificial enzyme scaffold.
• To create a stable 6HB structure with predictable side-chain group positioning and orientation for cooperative catalysis.
• To investigate the enzyme-like hydrolytic activity of engineered 6HB peptides.

Main Methods:

• Designed a novel 6HB scaffold using modified C34 and N36 peptides.
• Introduced histidine residues into the 6HB structure to create catalytic sites.
• Engineered the solvent-exposed face with Glu(-)-Lys(+) salt bridges to enhance stability and helicity.

Main Results:

• The histidine-modified 6HB C34(H13/20)/N36(H15/22) exhibited enzyme-like hydrolytic activity towards p-nitrophenyl acetate (PNPA), with a catalytic efficiency (k(cat)/K(M)) of 3.66 M⁻¹s⁻¹.
• Catalytic activity was modulated by factors affecting 6HB assembly, such as HIV fusion inhibitors and denaturants.
• Stabilization with Glu(-)-Lys(+) salt bridges increased 6HB helicity, stability, and catalytic efficiency to 6.30 M⁻¹s⁻¹.

Conclusions:

• A unique 6HB system was successfully assembled, providing a stable scaffold for artificial enzyme design.
• The engineered 6HB demonstrated cooperative catalysis through imidazole groups, mimicking natural enzyme function.
• This 6HB scaffold can be further engineered to mimic the function of enzymes and other biomolecules.