Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

Abstract

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a β-glycosidase enzyme (β-gly) and W492G in a β-glucuronidase enzyme (β-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan sidechain that serves as a buttress for the active site architecture. In both cases we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through detailed analysis of β-gly W33G kinetics we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of β-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate β-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

“…Several lid type (␤/␣) 8 barrel proteins studied to date use a loop extension within the ␣/␤ barrel region, and within the GH1 family, small changes to loops in the position of loop A have been shown to allow allosteric control (50). Also similar to other lid domains, both loop A and a small region of the C terminus may be intrinsically disordered (Fig.…”

“…27N is truncated at the N terminus of SFR2, with residue 27 being the first residue present from the original SFR2 sequence. Loop 1 and loop 2 constructs replace loop A (residues 67-157) with the equivalent loop from S. solfataricus (loop 1) or with a known ␤-turn from an artificially constructed (␤/␣) 8 (50). All DNA products were sequenced at the MSU RTSF facility and shown to be correct prior to protein production.…”

“…Either the equivalent loop from S. solfataricus GH1 (residues 14 -64) was substituted (loop 1) or the artificially designed ␤-turn KQFARH, with only a structural role (49), was substituted (loop 2). Note that the S. solfataricus GH1 loop was not wild type but included a mutation shown to allow allosteric control by indole (50). Mutant constructs and wild-type SFR2 were expressed in yeast producing the substrate MGDG, and the resulting lipid profile was examined by thin layer chromatography (Fig.…”

Engineering Biomolecular Switches for Dynamic Metabolic Control

“…This strategy has recently been demonstrated by designing a de novo allosteric effector site directly into the Engineering Biomolecular Switches for Dynamic Metabolic Control catalytic domain of an enzyme and it is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. In the two examples given by Deckert and coworkers [50], W33G in a β-glycosidase enzyme and W492G in a β-glucuronidase enzyme, indole-dependent activities were engineered into enzymes by removing a buried tryptophan side chain which served as a buttress for the active site architecture. In both cases the loss of function can be restored by the subsequent addition of indole.…”

Reversible Ca2+ Switch of An Engineered Allosteric Antioxidant Selenoenzyme

“…Among these successful examples, the progress of naturally engineered allosteric proteins employed to dynamically modulate enzyme activity is still relatively slow. [6] The major challenges of this strategy include not only grafting new catalytic moieties (e.g., catalytic and recognition components) into an appropriate position within a substrate-binding cleft, but also utilizing the allosteric conformational change to precisely control the designed protein function.…”