Novel inter-protein cross-link identified in the GGH-ecotin D137Y dimer

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Biology

Background:

• The Ni(II) complex of glycyl-glycyl-histidine (GGH) facilitates oxidative protein cross-linking.
• Site-specific cross-linking is crucial for analyzing protein-protein interactions in complex biological mixtures.
• Previous work suggested tyrosine residues are the primary targets for this cross-linking reaction.

Purpose of the Study:

• To perform a complete structural analysis of a cross-linked GGH-ecotin dimer engineered with a tyrosine at the protein interface.
• To characterize the specific cross-linking site and reaction mechanism.
• To assess the general utility of the GGH cross-linking system.

Main Methods:

• Mass spectrometry
• Chemical derivatization methods
• Structural analysis of cross-linked GGH-ecotin D137Y dimer

Main Results:

• A single, novel cross-link was identified between N-terminal glycine residues and engineered tyrosine 137.
• The cross-link was localized to a single site without other protein modifications.
• A reaction mechanism involving dopaquinone derived from tyrosine and the protein backbone was proposed.

Conclusions:

• High protein cross-linking yields can be achieved without requiring two tyrosine residues in close proximity.
• The GGH cross-linking reagent demonstrates broader utility than previously assumed.
• This system provides a powerful tool for mapping protein-protein contacts.