Design of a switchable eliminase

Abstract

The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.protein design | catalysis | pKa T he design of enzyme-like catalytic proteins is a challenging endeavour that has been approached using combinatorial (1) and computational strategies (2), as well as by following simple chemical principles (3, 4). The Kemp elimination (5) (Scheme 1) is an extensively studied benchmark for catalyst design (6-8) as a model for enzymatic C-H bond abstraction. Previously, computational methods were employed to alter the active sites of natural enzymes to catalyze this reaction (7). Mutation of a dozen or more side chains resulted in catalysts that showed rate enhancements of 10 4 to 10 5 relative to a given reference rate. Here, by combining a few physical principles with modern computational tools, we show that a single mutation can confer similar catalytic efficiency into a much simpler and allosterically switchable scaffold that previously had no catalytic activity. This approach circumvents often contentious questions concerning residual activities of the enzymatic framework or the appropriate reference background rate for expression of a "rate enhancement" while also suggesting a practical approach to the design of catalytically amplified sensors.Here we applied a minimalist approach to protein design (9-11), which emphasizes the use of the simple scaffolds and the min...

Iterative approach to computational enzyme design

“…1), a well-studied model system for the deprotonation of carbon (13). The KE was selected as a model reaction for this study because catalysts for it have been reported in multiple protein scaffolds (2,(14)(15)(16). In addition, from a computational design perspective, the use of the KE allows a direct comparison to the eight enzymes that were computationally designed for this reaction by Röthlisberger et al (2).…”

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

“…computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2)(3)(4)(5)(6)(7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig.…”

Rational design of a ligand-controlled protein conformational switch

“…These approaches are based on finding an optimal sequence for a given single structure or ensemble of related states, and do not provide a strategy to construct a protein capable of large on-demand conformational transitions (4, 5). A number of multistate protein design algorithms (4, 6) have been proposed; however, designing an experimentally confirmed, regulatable multistate protein, or a conformational switch (5), still remains as a challenging task because of the necessity of engineering and controlling multiple protein states (4,7,8).…”

“…These approaches are based on finding an optimal sequence for a given single structure or ensemble of related states, and do not provide a strategy to construct a protein capable of large on-demand conformational transitions (4, 5). A number of multistate protein design algorithms (4, 6) have been proposed; however, designing an experimentally confirmed, regulatable multistate protein, or a conformational switch (5), still remains as a challenging task because of the necessity of engineering and controlling multiple protein states (4,7,8).Such a conformational switch protein has great advantages in cell signaling, because it can be used as a universal regulatory domain (9) for precise, specific, and temporal control over rapidly activated signaling proteins (5, 10-15). Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5, 13).…”