The Next Frontier for Designing Switchable Proteins: Rational Enhancement of Kinetics

Abstract

Designing proteins that can switch between active (ON) and inactive (OFF) conformations in response to signals such as ligand binding and incident light has been a tantalizing endeavor in protein engineering for over a decade. While such designs have yielded novel biosensors, therapeutic agents, and smart biomaterials, the response times (times for switching ON and OFF) of many switches have been too slow to be of practical use. Among the defining properties of such switches, the kinetics of switching has been the most challenging to optimize. This is largely due to the difficulty of characterizing the structures of transient states, which are required for manipulating the height of the effective free energy barrier between the ON and OFF states. We share our perspective of the most promising new experimental and computational strategies over the past several years for tackling this next frontier for designing switchable proteins.

Figure 1.

Manipulating barrier heights to accelerate switching rates. (A) Free energy diagram of a protein biosensor with stable OFF and ON states, showing the transition path between them (red). Optimizing the equilibrium properties of the switch (turn-on/turn-off ratio, limit of detection) can be achieved by introducing mutations that shift the relative thermodynamic stabilities of OFF and ON conformations and alter the affinity of the ON state for the target ligand. (B) As in a classic enzyme mechanism, conformational switching can be accelerated by stabilizing the TSE. (C) In practice, it is often more tractable to lower the TSE barrier by destabilizing the ON and OFF folds by introducing mutations that delete interactions that are present in the stable states and absent in the TSE.

Figure 2.

Protein conformational switches and their response times. (A) The calbindin-AFF construct (PDB ID for calbindin D_9k: 3ICB) switches via Ca²⁺-driven unfolding/folding of two duplicate EF-hands (cyan and magenta) and their dissociation/docking with a shared region (gray). (B) The GCaMP calcium sensor (PDB ID: 3EK4) entails Ca²⁺-induced binding of CaM (cyan) to the RS20 peptide (magenta), which protects the GFP (gray) chromophore from solvent and turns on fluorescence. (C) The LOVTRAP optogenetic construct (PDB ID: 5EFW) involves light-triggered dissociation of the Jα helix (magenta) from LOV2 (gray), resulting in dissociation of Zdark (cyan). (D) The lucCage biosensor (PDB ID: 7CBC) is composed of a cage (gray) and a latch (cyan), to which an analyte recognition domain (magenta) has been fused. Binding of the analyte together with a key (which resembles the latch; not shown) causes the latch to dissociate and expose a sequence in the latch that complements and activates a reporter enzyme. (E) The SARS-CoV-2 spike protein (PDB ID: 6VXX) involves opening of the receptor binding domain (cyan) from the core domain (gray), as gated by a glycan (magenta) attached to the N343 residue.

Figure 3.

Weighted ensemble simulations of the opening of the SARS-CoV-2 spike protein. (A) Schematic of the weighted ensemble strategy. Trajectories (blue circles) are initiated from state A with equal statistical weights, propagating the dynamics in parallel (blue arrows) for fixed time intervals and applying a resampling procedure after each time interval to ensure equal coverage of configurational space (in this illustration, two trajectories per bin along a two-dimensional progress). The resampling procedure involves replicating trajectories that make transitions to less visited bins and occasionally terminating trajectories that have not made such transitions while rigorously tracking the trajectory weights (indicated by the sizes of the circles). The process of running dynamics and resampling is repeated until a desired number of trajectories have arrived in the target state B. (B) The SARS-CoV-2 spike activation process simulated using the WE strategy. The simulations involved the head region of the spike protein (gray) with full glycosylation (blue) and captured hundreds of switching pathways from the “down” state of the receptor binding domain (cyan) to the “up” and “open” states. Based on these pathways, the glycan attached to the N343 residue (magenta) functions as a gate that controls the switching process.