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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.
In the secretory pathway, many secreted proteins or transmembrane proteins are initially synthesized as protein precursors and subsequently cleaved by proprotein convertases before they become fully functional. The cleavage of protein precursors by proprotein convertases is involved in the molecular pathogenesis of a wide range of human diseases including cancer, neurological disorders, autoimmune diseases and various viral infections1. So far, seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1/PC3/SPC3, PC2/SPC2, PC4/SPC5, PACE4/SPC4, PC5/PC6/SPC6 and PC7/PC8/SPC7/LPC. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar2,3. Among the seven mammalian proprotein convertases, the cleavage of furin is the best characterized. The 3D crystal structure of dec-RVKR-cmk inhibited mouse furin binding pocket has been solved by Henrich and co-workers4. Furthermore, in authors' recent study, instead of a canonical four-residue motif R-X-[K/R]-R↓, the furin cleavage site has been characterized as a 20-residue motif that can be divided into two parts with distinct physical properties: 1) a core region (8 amino acids, position P6-P2´) inside the furin binding pocket that contributes to the binding strength of the substrate and 2) two polar regions (8 amino acids, positions P7-P14; and 4 amino acids, positions P3´-P6´) outside the furin binding pocket that mainly contribute to the solvent accessibility of the substrate5.
The 3D structure of the furin binding pocket4 and the physical properties of the core region P6-P2´ of the furin cleavage site motif5 provide a template with which to compare the local structural-functional relationship of all mammalian proprotein convertases. In this report, a multiple sequence alignment of mammalian proprotein convertases was generated, and the physical properties of mutations in the binding pockets were examined. The functional consequences of mutations were interpreted using the 3D structure. The results indicated two residues that play a key role in regulation of the substrate specificity of mammalian proprotein convertases. This finding provides insights into the molecular evolution of the enzyme function of the proprotein convertase family. The comparison of 3D structures also suggested a novel design for specific small molecular inhibitors that will aid the study of the functional redundancy of mammalian proprotein convertases and the development of therapeutic applications.
The eight amino acids of the core region P6-P2´ of a furin substrate are located inside the furin binding pocket and are the main factor determining the binding strength of a substrate5. The residues of the mouse furin binding pocket surrounding the core region P6-P2´ of furin substrates was analyzed in the 3D crystal structure of the dec-RVKR-cmk inhibited mouse furin binding pocket4 and also in the authors' recent study on the furin cleavage site motif5. Table Table11 lists the structural environment surrounding the substrate positions P6-P2´.
To study the substrate specificity of the other six proprotein convertases (PC1, PC2, PC4, PACE4, PC5 and PC7), the 3D structural environments provided by these six different mammalian proprotein convertases were compared against furin. A multiple sequence alignment of the binding pockets of seven mammalian proprotein convertases was generated using Clustalx6 and are publicly available on the associated website www.nuolan.org. The residue positions (listed in Table Table1)1) of the binding pockets surrounding the core region P6-P2´ were examined in the multiple sequence alignment and mutations were identified (Figure (Figure1)1) (Table (Table22).
Generally, changes in amino acids at specific positions in the binding pockets can be classified into three types:
It is striking that mutations in Glu230 or Glu257 were repeatedly observed, these being key residues interacting with substrate positions P4-P6 (Figure (Figure2).2). Two of the most notable features and interactions at substrate positions P4-P6 have been observed in previous experiments2,3,7-9 and can be explained by the 3D structure in this region:
It should be emphasized that what compensates for the loss of the positive charge at substrate position P4 is not the observed presence of a positive charge at substrate position P5 or P6, but the interaction force between the positive charge and negative charge formed at substrate position P5 or P6. Therefore, in order to maintain the sensitivity and compensatory effect of the positive charge at the substrate positions P4-P6, negatively charged residues are required in the binding pocket. Mutations in Glu230 or Glu257 change the overall negatively charged density of the binding pockets of the other six mammalian proprotein convertases, with profound functional consequences:
Although these conclusions were entirely derived from the theoretical 3D structure study, they are supported by existing experimental data. The cleavage site of integrin alpha 4 precursor (HVISKR597↓, GI_124945) lacks a positively charged residue at position P4, and compensates for this loss with a positively charged histidine at position P6 that is protonated in the acidic secretory compartments. By co-infecting furin-deficient LOVO cells with individual proprotein convertases and measuring immunoprecipitated products of cleavage, Bergeron and co-workers showed that processing of integrin alpha 4 precursor is performed efficiently by furin, PACE4 and PC5 (similar specificity), and much less efficiently by PC1 (reduced sensitivity for positive charge at position P6), and not by PC710. In another study, Hendy and co-workers co-expressed pro-parathyroid hormone and proprotein convertases in BSC-40 and GH4C1 cells. The mass spectrometric analysis indicated that the cleavage site of pro-parathyroid hormone (KSVKKR31↓, GI_4506267) can be 2-4-fold more efficiently cleaved by furin than PC1 and PC211. Again, the pattern KSVKKR↓ is characterized by the efficient compensation for the absence of a positive charge at position P4 by the presence of a positive charge at P6, which is preserved for furin, but reduced for PC1 and PC2.
To summarize, a negatively charged density at position 230 and 257 is a key factor that regulates the substrate specificity of mammalian proprotein convertases. In the course of molecular evolution, the enzyme function of the mammalian proprotein convertase family seems to have been fine-tuned by adjusting the density of the negative charge of the binding pockets. While this mechanism is supported by both the theoretical approach and experimental data, it is not the only structural mechanism. Which amino acid is present at other substrate positions such as P1' and P2' and the solvent accessibility of the substrates also affect the cleavage efficiency. In addition, physical interaction between the substrates and binding pockets of proprotein convertases is not the only way in which cleavage is regulated; the function of proprotein convertases is also regulated at other levels such as the level of expression in different tissues, their sub-cellular localizations, and the acidity of the different secretory compartments.
Cleavage of proprotein convertases can be efficiently inhibited by a small molecule dec-RVKR-cmk 18,19. The structure of dec-RVKR-cmk is characterized by positively charged residues at substrate positions P1, P2 and P4. Consequently, this small molecule dec-RVKR-cmk efficiently binds to all seven mammalian proprotein convertases, and only has limited use for studying the functional redundancy of mammalian proprotein convertases.
Specific short peptide inhibitors for only a specific group of proprotein convertases will aid the study of the functional redundancy of proprotein convertases. The results from a comparative study of the 3D structure immediately suggested a novel pattern for small molecule inhibitors targeting a specific group of mammalian proprotein convertases. The general 3D structure of this small molecule is similar to that of short peptides [K/R]-X-V-X-K-R, which omit a positively charged residue at position P4, but add a positively charged residue at position P6. The loss of the negative charge on Glu230 and Glu257 of the PC1 and PC2 binding pocket likely results in reduced sensitivity to the positive charge at substrate positions P5 and P6, whereas at least one negative charge is conserved at Glu230 or Glu257 in the binding pockets of the other five mammalian proprotein convertases: furin, PACE4, PC5, PC4 and PC7. Therefore, a short peptide [K/R]-X-V-X-K-R, with no positive charge at position P4 and one positive charge at position P6, should have a higher affinity to furin, PACE4, PC5, PC4 and PC7 than to PC1 and PC2.
Interestingly, in the course of molecular evolution, PC1 and PC2 have acquired very different tissue distributions from the other five mammalian proprotein convertases2: PC1 and PC2 are highly expressed in neuroendocrine tissue; PC4 is highly expressed in the testicular and ovarian tissues; furin, PACE4, PC5 and PC7 are widely expressed. Given the different expression levels of the different mammalian proprotein convertases in the various tissue types, a small specific molecular inhibitor specifically targeting furin, PACE4, PC5, PC4 and PC7 would be especially interesting for the development of therapeutic applications. Various derivatives of this novel short peptide pattern [K/R]-X-V-X-K-R need to be tested for the optimal binding affinity and specificity, and further work is needed to confirm their potential.
The 3D crystal structure of the dec-RVKR-cmk inhibited mouse furin binding pocket was retrieved from the Protein Data Bank ID:IP8J4. To analyse the 3D structure of the active site and calculate hydrogen bonds of molecular interactions, Swiss-PDB Viewer17 was used and the procedures described by Guex et al. on the Swiss-Pdb Viewer site http://www.expasy.org/spdbv/ were followed.
The mammalian orthologues of seven proprotein convertases (furin, PC1/PC3/SPC3, PC2/SPC2, PC4/SPC5, PACE4/SPC4, PC5/PC6/SPC6 and PC7/PC8/SPC7/LPC) were identified by the BLAST algorithm20. Five mammal species with relatively well-assembled genomes were selected: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus and Canis familiaris. A multiple sequence alignment was produced by Clustalx6.
Our results extended our previous study on the physical properties of a 20-residue furin cleavage motif by determining the functional consequences of mutations at two specific positions (230 and 257) of the binding pockets of the other six mammalian proprotein convertases. We showed that the residues at positions 230 and 257 are frequently mutated in the binding pocket of mammalian proprotein convertases, resulting in a change in the density of the negative charge of the binding pockets. Comparison of the 3D structures suggests that different proprotein convertases have different sensitivities for the positive charge at substrate positions P5 and P6. Specifically, PC1 and PC2 may have reduced sensitivity for positively charged residues at the substrate position P5 or P6, whereas the substrate specificities of furin, PACE4, and PC5 may be very similar to each other. The density of the negative charge of the binding pockets seems to have played a key role in the molecular evolution of the enzyme function of the mammalian proprotein convertase family. This may allow the rational design of novel specific molecular inhibitors targeting specific members of the mammalian proprotein convertase family.
This project was started as Sun Tian's coursework for 445.039 Projekt Medizinische Informatik und Neuroinformatik at TUGraz and was fund by the GENAU Bioinformatics Integration Network PhD programme until June 2007 (PhD programme and grant leader: Professor Zlatko Trajanoski at TUGraz) and NFSC grants 10372118 (JW). Sun Tian gratefully acknowledges the support of Professor Zlatko Trajanoski.