Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection

Abstract

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 highresolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.C aspase function in cell development and cell death results from a continuum of enzyme activity, in which an as-yetundefined activity threshold is required for cell death. At subthreshold levels, caspase activity is important for a variety of physiological reactions (referred to as adaptive responses), including remodeling the cytoplasm (1), cell differentiation (2), neuron pruning (3), receptor endocytosis (4), macrophage function (5), and development of the eye lens (6) and inner ear (7). The roles of caspases in apoptosis are well known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation.Cells use two general mechanisms to modify caspase activity, through modulating levels of active caspase or through allosteric mechanisms that change the distribution of conformations in the native ensemble, although the two are not mutually exclusive. Levels of caspase-3 are controlled by cleavage of the inactive zymogen to yield a dimer of protomers (Fig. 1A) (8,9), and this process is responsive to several signaling pathways, such as transient expression of the Bad-Bax cascade (10) or phosphorylation of the zymogen (Fig. 1B) (11). Alternatively, inhibitor of apoptosis proteins (IAPs) affect levels of active caspase-3 by dire...

DRoP: Automated detection of conserved solvent‐binding sites on proteins

“…By coloring amino acid residues according to the conservation ranking of the water molecules with which they interact, the DRoP color feature allows the direct visualization of water‐mediated networks connected across the protein molecule. This feature was previously used to identify allosteric connections in Ras GTPase and in caspase 3, and is illustrated here with RNase A where the N‐terminal α1 network published by Zegers et al was identified with an additional extension linking the active site to the hinge region through α3. This network is composed of water molecules that are highly conserved regardless of crystal contacts.…”

“…Water molecules are important for numerous aspects of protein folding, structure of the native state, protein dynamics and function, and binding events . Analysis of crystallographic water molecules positioned on the protein surface has revealed water‐mediated allosteric networks linking distant parts of the structure that on first sight do not seem to be connected . The Protein Data Bank (PDB) is a searchable database with various multiple high‐resolution X‐ray structures of proteins in different states, providing a rich data set of structures for analysis of protein‐water interactions.…”

“…4,5 Analysis of crystallographic water molecules positioned on the protein surface has revealed watermediated allosteric networks linking distant parts of the structure that on first sight do not seem to be connected. [6][7][8][9][10][11][12] The Protein Data Bank (PDB) 13 is a searchable database with various multiple high-resolution X-ray structures of proteins in different states, providing a rich data set of structures for analysis of protein-water interactions. In addition to containing a large number of individual structures, the PDB has benefited from advances in structure-based ligand design, where small molecules serve as fragments of a larger ligand.…”

“…6,9,[15][16][17][18] Superposition of multiple structures of a given protein in different crystallographic environments and/or solvent conditions results in large data sets, with careful analysis revealing sites where organic molecules or crystallographic water molecules cluster consistently and can bridge secondary structures across the protein surface. 6,8,19 This type of work has generally been done by visual inspection in a laborious process that only few researchers have been willing to tackle. Therefore, the vast amount of data contained in multiple structures of a given protein in the PDB have been largely unexplored.…”

Modifications to a common phosphorylation network provide individualized control in caspases

“…One allosteric site of interest, S150, is located in a loop at the C-terminal end of helix-3 near the dimer interface, and this serine residue (or threonine in caspase-7) is conserved in all human caspases except caspases-10 and -14 (4). Helix-3 is an important regulator of effector caspase activity because fluctuations in the Nterminal region of the helix disrupt conserved water networks in caspase-3 and reposition the catalytic cysteine and histidine (24,25). In addition, the same region of helix-3 and the adjoining β-strands undergo a coil-to-helix transition in caspase-6, which also disrupts the catalytic residues by extending helix-3 (22).…”