Here, we report the energetic, structural and sequence requirements for cleavage of ENaC by CAP3 and furin. While we focus our attention on activation of ENaC by proteases, the results of our computational analyses are broadly applicable to catalysis by serine proteases. Limited endoproteolysis of ENaC is a complex mechanism for regulating Na+
absorption by epithelia. Numerous proteases that stimulate ENaC have been identified, alongside endogenous protease inhibitors that oppose this effect (12
). Thus, ENaC may be controlled in a tissue specific manner by the complement of endogenous proteases and anti-proteinases expressed in a given cell type. We experimentally characterized the effects of CAP3 co-expression on proteolysis and stimulation of ENaC in Xenopus
oocytes. We identified two regions of γ-ENaC susceptible to direct cleavage by CAP3. Cleavage after any of several basic residues within each region is necessary and sufficient for full stimulation of ENaC. These observations support three main conclusions. First, cleavage of the γ-subunit alone accounts for CAP3 stimulation of ENaC. Second, though limited in extent, the specificity of γ-ENaC cleavage is not based on stringent docking of extended segments of γ-ENaC within the active site in CAP3. Instead, multiple basic residues comprising the γ-ENaC furin site, along with those including and flanking 178-RKRK are exposed for proteolysis. Finally, CAP3 alone can achieve full proteolytic stimulation of ENaC.
Structural studies of ENaC are set back due to the unavailability of a complete atomistic structure of the heteromultimer. Recently, different ENaC subunits were threaded onto the structure of a homologous acid-sensing ion channel (ASIC1) (44
). In the sequence alignment used for model building, the region in γ-ENaC containing the putative cleavage sites for CAP3 and furin was designated the “hypervariable region” (44
). Consequently, the structural features of this region are unavailable despite the modeling efforts. The observation that the protease cleavage sites in ENaC are intrinsically disordered is in line with the proposed structural requirement for serine protease activity (5
). To study the energetic basis for substrate recognition by CAP3 and furin, we performed DMD simulations of both enzymes with different candidate peptides. Although crystal structures of matriptase/CAP3 (PDB: 1EAW) and furin (PDB: 1P8J) with bound inhibitors are available for this effort (30
), we reasoned that the static orientation of an inhibitor in the catalytic pocket does not effectively capture the dynamics of enzyme-substrate complexes. Therefore, we resorted to DMD simulations and we observed from the low energy configurations that a charge complementary pocket in both furin and CAP3 mediates critical enzyme-peptide interactions (). While these simulations are not intended to model the hydrolysis reaction carried out by either enzyme, they provide insight into the binding properties of a given enzyme-peptide combination. Given that substrate recognition is the first step in hydrolysis, we consider a peptide that can reach the vicinity of the enzyme (3–5 Å) as a potential substrate for cleavage. By maintaining most of the enzyme static during simulations, we assume that furin and matriptase do not undergo significant conformational changes during peptide recognition. Furthermore, by using an implicit solvent model (EEF1) for treating solvent interactions, we disregard specific water-mediated hydrogen bond formation involved in hydrolysis. Therefore, our simulation studies are only aimed at determining whether a given peptide is energetically suitable for presenting itself in the proximity (within 3–5 Å) of the active site of furin or matriptase, and not to quantify the rate of hydrolysis of a given peptide by a protease. PMF analysis with Seq1 features low energy basins at the active sites of both furin and CAP3 suggesting that Seq1 can be a substrate for both enzymes. Interestingly, analogous computational study of Seq3 indicates that 132KESR can be a substrate for CAP3 but not for furin. This result is consistent with our observation that CAP3 cleaves and stimulates ENaC effectively when 132KESR are the only potentially susceptible residues available near the furin site, whereas exogenous furin is much less effective at stimulating ENaC in this context (). These computational analyses of WT sequences, with our functional and biochemical analyses of WT and mutant γ-ENaC clearly indicate that the γ-ENaC sequence 132-KESRKRR is highly susceptible to cleavage by CAP3.
CAP3, unlike furin in this study, or CAP2 in our previous work (14
), appears to cleave γ-ENaC after several basic residues within 132-KESRKRR, including 135R, 136K and 138R. Such greater susceptibility of γ-ENaC to cleavage may reflect CAP3’s low substrate stringency coupled with favorable substrate access due to local structural disorder. 2D-PMF analyses of all peptide-catalytic site combinations considered in this study broadly support this notion. Energy minima at 3–5 Å indicate close contact between peptide and catalytic site required for proteolysis (), whereas low energy basins beyond 5 Å are suggestive of electrostatic attraction between the net positively charged peptides and the negatively charged surface loops of the enzyme. The absence of a low energy basin at a distance of 3–5 Å from the active site reflects the fact that Seq3 is a less suitable substrate for furin than CAP3 (). Seq1 and Seq2 feature energy minima at 3–5 Å from the active site of both CAP3 and furin (compare ). Low energy basins corresponding to interaction between the peptide and the surface loops of the enzyme are observed beyond 5 Å for all peptides in simulations. However, only Seq1 and Seq2 are energetically compatible to be in the proximity of the active of CAP3 and furin, as evidenced from the PMF analysis (). PMF analysis indicates that it is favorable for Seq3 to be in the vicinity of CAP3, but not furin. In light of these observations, we propose an “anglerfish” mechanism analogous to the characteristic mode of predation by the anglerfish where the fish represents the enzyme, with its illicium being the surface loops that capture the peptide to be cleaved and proteolysis being the eventual irreversible event (). The realization of this mechanism is confined by the assumption that proteolysis is guaranteed if a peptide is energetically compatible to be in the vicinity (3–5 Å) of the active site of a hydrolytic enzyme.
“Anglerfish” mechanism of peptide recognition and cleavage by MAPs
Our examination of CAP3 activity suggests that several different events underlie stimulation of ENaC. Mutation analysis suggests that 132K participates in a sequence (132-KESR) predicted to be optimal for cleavage by CAP3 (32
). Residual cleavage and stimulation of ENaC containing γ-135-RQQQ requires the intact sequence 132-KESR. Mutagenesis of the furin sites in α-ENaC has no effect on CAP3 stimulation of ENaC, even though CAP3 converts all FL WT α-ENaC to fragments expected from cleavage at the two furin sites, similar to our previous study with CAP2 (14
). The stimulation of ENaC by CAP1 was also linked by mutagenesis to γ-ENaC (16
). Complete proteolytic stimulation of ENaC by soluble serine proteases, including trypsin, hNE and plasmin is also exerted by cleavage of the γ-subunit (40
). Thus, our results demonstrating that CAP3-mediated stimulation of ENaC can be blocked by mutations in γ-ENaC add to a growing body of data indicating that γ-ENaC plays a central role in the proteolytic regulation of ENaC.
Based on our results, cleavage of γ-ENaC mediated by CAP3 occurs in two regions: the first is discretely defined by the furin site and the second includes basic residues centered on and surrounding the 178-RKRK tract. CAP3’s low stringency requirements for cleavage are consistent with its cleavage of γ-ENaC at multiple basic residues downstream of the furin cleavage site. Our results provide a compelling hypothesis that recognition and/or cleavage of γ-ENaC by proteases is substantially determined by accessibility of key sites rather than solely by a stringent structural requirement. The ability of certain proteases to activate ENaC has been linked to a specific residue or tract within 172–202 (16
). Based on such work for prostasin (CAP1), the initial expectation was that CAP3 would cleave at the 178-RKRK tract. However, when 178-RKRK is unavailable, both CAP2 and CAP3 readily cleave at multiple basic residues in the region 172–202. This phenomenon is consistent with the concept that this region is highly accessible to proteases due to intrinsic structural disorder. The low stringency of CAP3 explains why we could not identify a single residue or short tract that can be mutated to block CAP3 stimulation of ENaC. We expect the accessibility of either region to increase with time and extent of proteolysis, leading to further cleavage and consequent activation by relatively non-specific proteases.
Our results indicate that CAP3 potently stimulates ENaC in an in vitro
setting, which is dependent on direct catalytic attack of the protease on extracellular segments of γ-ENaC. It has been suggested that CAP3 completes its auto-activation at the cell surface (49
). As a step towards understanding the colocalization of ENaC and CAP3 in vivo
, we performed immunofluorescence microscopy to confirm that matriptase localizes to the subapical pools near the plasma membrane of human bronchial epithelial (HBE) cells derived from different individuals (Figure S5
). ENaC can be detected in apical and subapical compartments of HBE cultures and is processed by limited cleavage in these cells (51
). Thus, it appears highly likely that ENaC is cleaved by matriptase in HBE cells. Additional work is needed to confirm that ENaC and active CAP3 occupy positions at the cell surface that enable ENaC cleavage, but our results clearly imply that CAP3 is capable of activating ENaC independent of furinlike convertases. CAP3 also activates CAP1 (53
), which stimulates ENaC regardless of its catalytic activity (54
). These and other recent observations suggest a possibility for a cascade of interactions at the cell surface involving CAP3 and other proteases like CAP1, exercising dynamic control over proteolytic regulation of ENaC (55
We conclude that CAP3, a MAP, can independently activate ENaC without contributions from furinlike convertases or soluble proteases, without a requirement for specific structural motifs for recognition and/or cleavage. Our computational analyses give rise to a potentially generic “anglerfish” mechanism applicable broadly to serine protease mediated recognition and cleavage.