Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

Abstract

Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2' inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2' of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1) mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2) two proprotein convertases (PC1 and PC2) may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5) are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7) compared to the remaining two (PC1 and PC2). The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein convertases and the development of therapeutic applications.

Keywords: evolution of gene family.; mammalian proprotein convertases; small molecular inhibitor; substrate specificity.

Figure 1

Mutations in the key residues in the binding pockets of mammalian proprotein convertases Black indicates conserved residues. Blue indicates that a mutation changes the amino acid type but does not significantly affect the physical properties of the side chain of an amino acid. Red indicates that a mutation affects the physical properties of the side chain of an amino acid. The multiple sequence alignment was generated with Clustalx.

Figure 1

Mutations in the key residues in the binding pockets of mammalian proprotein convertases Black indicates conserved residues. Blue indicates that a mutation changes the amino acid type but does not significantly affect the physical properties of the side chain of an amino acid. Red indicates that a mutation affects the physical properties of the side chain of an amino acid. The multiple sequence alignment was generated with Clustalx.

Figure 2

The sensitivity and the compensatory effect of the positive charge at substrate positions P4P6 require negatively charged residues in the binding pocket of mammalian proprotein convertases. At position P4, the positively charged residue arginine interacts with Glu236, Asp264, and Tyr308 of the furin binding pocket. Hydrogen bonds of molecular interactions at position P4 are calculated and shown as green lines. If a positively charged residue is absent at position P4 and favourable interactions with negatively charged Glu236 and Asp264 of the furin binding pocket are lost, this loss can be compensated for by the gain of an interaction between the negatively charged Glu230 and Glu257 of the binding pocket and a positively charged residue at substrate position P5 or P6. The distance between Glu230 and Glu257 is only 16.4Å and facilitates flexible interactions in this region. The density of the negative charge in the binding pocket is important for maintaining the sensitivity and the compensatory effect of the positive charge at substrate positions P4-P6. This structure was modeled based on the published 3D structure of the furin catalytic domain in complex with an inhibitor (Protein Data Bank ID: IP8J). The figure was generated with Swiss-Pdb Viewer and was modified from the authors' previous publication.