Novel inter-protein cross-link identified in the GGH-ecotin D137Y dimer.
In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.
Person
M D
MD
Department of Pharmaceutical Chemistry, University of California at San Francisco, 94143-0446, USA.
Brown
K C
KC
Mahrus
S
S
Craik
C S
CS
Burlingame
A L
AL
D001426:Bacterial Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D010455:Peptides; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C427216:glycyl-glycyl-histidyl-ecotin, recombinant; D009532:Nickel