Structure of an FCGBP Domain Confirms Hydrolysis at a GDPH Autocatalytic Cleavage Sequence

IgFC-binding protein (FCGBP) is a protein found in the protective mucus layer coating the intestinal epithelium. Human FCGBP has thirteen copies of a domain known as von Willebrand “D” (VWD). An intriguing feature found in eleven of the VWD domains is an autocatalytic cleavage site. The amino acid sequence Gly-Asp-Pro-His (GDPH) in these domains has been shown to undergo peptide bond hydrolysis due to the lability of Asp-Pro bonds. Cleavage at Asp-Pro bonds in vitro is accelerated by low pH and high temperatures, but FCGBP cleavage occurs under physiological conditions, perhaps promoted by the mildly acidic pH of the Golgi apparatus. The GDPH sequence and peptide bond hydrolysis are not conserved in many VWD domains of other proteins, indicating that this sequence is not required for folding of the domain and suggesting that cleavage has evolved for some other purpose. To visualize the cleavage site and begin to understand the structural and functional consequences of cleavage, we determined the crystallographic structure of an FCGBP VWD domain. The cleavage site is in a loop between the two first ß strands of the domain. The first strand is bound to the rest of the D assembly by a disulfide bond and buried in the ß sheet, such that cleavage does not dissociate the two fragments. However, cleavage appears to cause local conformational changes and the formation of new contacts. To characterize the mechanistic requirements for cleavage, we introduced point mutations at surrounding positions and analyzed their effect on GDPH cleavage.