Protein-peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase

Area of Science:

• Structural biology
• Biochemistry
• Molecular biology

Background:

• Linear motifs typically exhibit moderate binding affinity (Kd of ~0.1-10 µM) to shallow protein surfaces.
• Protein crystallization with linear motif peptides is difficult due to competing crystal packing forces.
• The extracellular signal-regulated kinase 2 (ERK2) binding site is prone to crystal packing interference.

Purpose of the Study:

• To develop a strategy for determining the crystal structures of protein-peptide complexes with weak binding affinities.
• To overcome challenges in crystallizing extracellular signal-regulated kinase 2 (ERK2) with docking peptides.

Main Methods:

• Rational surface engineering by mutating protein residues distant from the peptide-binding site to alanines.
• Utilizing alanine mutations to reduce non-specific crystal packing interactions.
• Structure determination of engineered ERK2 in complex with novel docking peptides.

Main Results:

• Surface mutations successfully decreased unwanted crystal packing of ERK2.
• The engineered ERK2 facilitated the structure determination of complexes with new docking peptides.
• This approach validated the utility of surface engineering in protein crystallography.

Conclusions:

• Protein surface engineering is a valuable strategy for overcoming crystallization challenges with weakly interacting partners.
• Negative selection in crystal engineering is critical for obtaining structures of protein-peptide complexes.
• The study provides a method for elucidating the structures of important biological complexes.