Homology modelling of the catalytic domain of human furin

Abstract

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca2+-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2', S3, S5 and S6 subsites may also contribute to substrate binding.Based on this model, protein engineering can be employed not only to test the predicted enzymesubstrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.

Mutations in the Catalytic Domain of Prohormone Convertase 2 Result in Decreased Binding to 7B2 and Loss of Inhibition with 7B2 C-terminal Peptide

“…The latter finding indicates that the residue Gln 242 and/or the residue Pro 243 play a critical role in the interaction of PC2 with 7B2. Based on the three-dimensional structural model proposed for furin (50) Both mutant PC2s, when expressed in AtT-20/27-kDa 7B2 cells, exhibit a slow rate of intracellular maturation and substantially reduced enzymatic activity in the conditioned medium as compared with wild-type PC2. Pro-PC2-(242-248) immunopurified from these cells also displays a highly reduced ability for autocatalytic conversion at acidic pH.…”

“…Released 7-amino-4-methylcoumarin was measured with a Fluoroscan Ascent fluorometer (Labsystems) using an excitation and an emission wavelength of 380 and 460 nm, respectively. The IC 50 values for the peptide inhibitors were calculated through nonlinear regression of the experimental data (fluorescence versus peptide concentration) using the program GraphPad (GraphPad Software, Inc., San Diego, CA).…”

“…It is noteworthy that both PC1s and furins contain a deletion in this sequence as compared with PC2s; the single G residue in PC1s and furins thus corresponds to the QP residues of PC2s (positions 242 and 243). The residues in this region (positions 242-248) are located within or in the vicinity of subsites S2-S5 of prohormone convertases, according to homology modeling of protein convertases based on the crystal structure of subtilisin (50,31). These residues could thus contribute to PC2-specific properties.…”

“…For this purpose, the primary sequences of several PC1, PC2, and furin enzymes from different species were aligned, and the residues within the predicted substrate binding sites (15,31,50) were compared. This analysis revealed that in positions 242-248 (numbering is that of PC2), PC2s contain the sequence QP(Y/F)MTD(L/I), which differs considerably from the corresponding sequence in PC1s and furins, G(I/E)VTDA (Fig.…”

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

“…These are precisely the two residues *156 and * 166 in subtilisin that were previously changed to Glu or Asp to generate specificity for positively charged PI residues (Wells et ai., 1987b). Modelling and engineering studies of proprotein convertases have shown that specificity for Arg(Pl) in this distinct subfamily of the subtilisin-like proteases is also dominated by an Asp(* 166) residue at the bottom of the S 1 pocket (Creemers et al, 1993;Siezen et al, 1994). The S4 pocket of NisP is predicted to be smaller than that of subtilisin due to several large predominantly hydrophobic side chains (Table II).…”