These mutational and structural studies begin to reveal the critical features of an extensive and conserved H-bonding network that couples the functional sites in caspase-1. The alanine-scanning experiments indicate that only four of the nine H-bonding side chains have a significant effect on activity, especially the Arg286-Glu390 interaction. These form a contiguous chain of interactions that connects the active and allosteric sites in the protease.
It is remarkable that so many of the H-bonding pairs seen in the on-state are not preserved in the off-state of caspase-1 (, ). From structural inspection alone, it is difficult to predict which of the residues involved in this extensive H-bonding network are most critical for enzyme activity. Neither the extent of H-bonding in one state nor the changes in H-bonding between on-state and off-state predict which residues should be most important (). Including bound water molecules in the H-bond inventory still does not improve predictive power (see Supplemental Table S1
). We conclude that changes in H-bonding patterns are not clear predictors of the effects on the activity we observe. In general, the substitutions had fairly small effects on the Hill coefficient. Even the 130- and 230-fold reductions in activity seen for the E390A and R286A caused only slight changes in the Hill coefficient, suggesting that cooperativity is a fairly robust property of the enzyme.
The effects of conservative substitutions in the critical Arg286-Glu390 salt bridge on both the structure and the function of the enzyme were somewhat surprising. One conservative shortening substitution (R286K) caused a 150-fold reduction in activity, whereas another one (E390D) had only a small 2-fold reduction.12
This substitution, combined with the R286K, caused partial restoration from the single R286K variant. The structures of the active forms of the enzyme, driven by reaction with the active-site titrant z-VAD-FMK, did not provide an obvious structural interpretation for the functional effects. For example, we see that the direct salt bridge is preserved in the dramatically reduced R286K variant; however, for the E390D variant, a water-mediated salt bridge is seen between Asp390 and Arg286. The most active variants are those that preserve both the salt bridge between positions 286 and 390 and the interaction with the water cluster. These latter interactions are lost in the R286K variant and preserved in the more active R286K/E390D variant.
Solvent accessibility calculations (Supplemental Table S2
) show that Arg286 in the wild-type structure is more solvent-exposed than Lys286 in the mutant variant. Thus, it is possible that the less solvent-accessible salt bridge in the mutant is energetically less favorable due to the internal environment of the protein not fully compensating for the desolvation of the salt bridge. We calculated the solvent-exposed polar and hydrophobic surface areas for the two salt bridge residues in each of the variants. Interestingly, we find that as the exposed hydrophobic surface area increases and the exposed polar surface area decreases, the activity of the variants also decreases. We are reluctant to make strong conclusions about the importance of specific H-bonding interactions with the water cluster based on this small data set. Nonetheless, we are impressed by the functional variation in these conservative substitutions.
Nonpreserving effects for conservative substitutions of electrostatic interactions in other systems have also been reported. Studies of Ras have demonstrated that the Gln61 position is sensitive to mutations that can reduce the kcat
of the RasGAP complex by at least 1000-fold. Interestingly, computational studies found that despite the direct electrostatic interactions this residue makes with other residues and substrates in the transition state, it is not directly involved chemically in GTP hydrolysis. Rather, this residue is crucial in helping to form the polar framework of the active site, and mutations of Gln61 disrupt the preorganized environment necessary for catalysis.16
The importance of electrostatic interactions has also been studied in the context of protein—protein interfaces. The structure of the p160 coactivator ACTR, in complex with the ACTR-binding domain of the CREB-binding protein CBP, identified a buried Arg-Asp salt bridge in the midst of a binding interface largely dominated by hydrophobic residues.17
Mutational studies of this salt bridge found that this interaction is especially important for specificity when discriminating between binding partners. Modification of this interaction by swapping the positions of the Arg and Asp residues to maintain the salt bridge abrogated binding.18
This observation is perhaps not surprising, as ion pairs reversal in stable local protein environments is generally destabilizing.19
These environments tend to form prepolarized sites organized to stabilize an ion pair of a given polarity such that swapping the residues in a salt bridge, as in the case of the ACTR—CBP binding interaction, will be destabilizing even when an electrostatic interaction is preserved. We have made perturbations to the electrostatic interaction in caspase-1 that we expected to be less disruptive than those in these other systems, yet we were surprised to find a significant impact on the catalytic activity in the R286K variants. It is possible that the reduction in kcat
seen with a R286K substitution is caused by disruption of a preorganized network of polar interactions essential for protease activity that involves not only the Arg286-Glu390 salt bridge but also surrounding interactions involving solvent water molecules.
The classic model of allosteric transitions predicts that proteins can adopt alternate conformations that are present in a preexisting equilibrium even in the absence of regulatory ligands.20
Recent studies in various systems have begun to provide direct experimental evidence for this theory and have extended it to allosteric regulation involving covalent modifications, in addition to ligand-binding events. NMR relaxation experiments on the nitrogen-regulatory protein C demonstrated that the activation of this protein by phosphorylation shifts a preexisting equilibrium between inactive and active conformations.21
In a similar vein, studies of the enzyme cyclophilin A found that the enzyme undergoes presampling of conformational states in the absence of substrate that mimics the dynamics seen during catalysis. Specific mutations cause shifts in the relative populations between conformations. In addition, cyclosporin A, which binds and inhibits cyclophilin A, shifts the enzyme to a single state. Thus, this drug works by locking the isomerase in one conformation.22
It is known that disruption of a salt bridge between the catalytic (Asp236) and regulatory (Lys143) subunits in Escherichia coli ATCase disrupts cooperative binding of the aspartate substrate.23,24
More recent work using small-angle X-ray scattering has found that disruption of this salt bridge by substitution with alanine results in an enzyme variant that exists in a reversible equilibrium between at least two states in the absence of ligands.25
This is in contrast to wild-type ATCase, which exists predominantly in a low-activity, low-affinity state. Therefore, disruption of a salt bridge in ATCase destabilizes one state, allowing the protein to exist in an equilibrium between two states in solution. In the case of caspase-1, the apo form of the enzyme crystallizes in the same conformation as the allosterically inhibited form. We believe that caspase-1, in solution, exists predominantly in an inactive conformation. The results presented here suggest that the active conformation, which is favored in the presence of an active-site ligand, is stabilized by the formation of the Arg286-Glu390 salt bridge. Disruption of this interaction could make it harder for the enzyme to visit the active conformation, leading to the decreased activity of the salt-bridge variants.
Overall, our studies point to a small set of side chains that form a contiguous circuit connecting the active and allosteric sites as being the most important for activity in caspase-1. Bioinformatic approaches analyzing coevolution of residues in large protein superfamilies show patterns of conservation and covariation suggestive of allosteric circuits. In these families, the majority of residues seem to evolve independently, while a small subset forms a linked network that is positioned for long-range communication through the structure.26
Mutational analysis at protein—protein interfaces reveals only a small subset of contact residues near the center of the binding interface drives the affinity of the interaction.8
Our alanine-scanning data on caspase-1 support a view that allostery is transmitted predominantly by a small subset of connected residues.