Design principles of protein switches

Abstract

Protein switches perform essential roles in many biological processes and are exciting targets for de novo protein design, which aims to produce proteins of arbitrary shape and functionality. However, the biophysical requirements for switch function - multiple conformational states, fine-tuned energetics, and stimuli-responsiveness - pose a formidable challenge for design by computation (or intuition). A variety of methods have been developed toward tackling this challenge, usually taking inspiration from the wealth of sequence and structural information available for naturally occurring protein switches. More recently, modular switches have been designed computationally, and new methods have emerged for sampling unexplored structure space, providing promising new avenues toward the generation of purpose-built switches and de novo signaling systems for cellular engineering.

Figure 1 |. What makes a protein switch?.

A, Protein switches couple inputs to functional outputs via structural responses. Due to the diversity of inputs and outputs, and the ability of variable structural responses to connect different stimuli and functions, protein switches are often envisioned as a “black box”. In contrast, as illustrated in B, engineering controllable switches de novo requires a specific and “designable” structural definition of the switch mechanism. Protein switch function arises from four essential components: the presence of multiple structural states, with distinct functions, that are differentially populated in response to an input, owing to their suitable energy landscapes. We illustrate these components with the natural example of calmodulin, a Ca²⁺-binding signaling protein.

Figure 2 |. Examples of switch mechanisms found in nature.

A, Local changes in geometry between two distinct conformations are commonly used as toggles for activity. B, Fold switching proteins involve exchange of entire secondary structure elements and can exhibit different functions specific for each state. C, Rearrangement of entire domains can occur via various mechanisms and regulate activity by increasing access to the domain and/or active site.

Figure 3 |. Designed protein switches.

A, Sequence-based switch design. These designs are heavily informed by homologous protein sequences to infer basic requirements for adopting one fold over another. This example [4] used sequence motifs of zinc finger proteins and helical bundles to design a Zn²⁺-dependent fold switch. B, Nature-inspired conformational switches. Switches in this category typically aim to emulate specific conformational changes seen in nature. Here, we depict a coiled-coil switch designed to mimic hemagglutinin, using Ca²⁺ binding as a trigger [11]. C, De novo designed modular protein switches. The LOCKR class of designed proteins [30] utilizes domain displacement as a switch mechanism and has been applied towards a number of different uses. D, Functionally coupled changes in geometry. By taking advantage of the knowledge of helical packing and metal binding motifs, the ROCKER protein [33] undergoes conformational exchange between different states, passing Zn²⁺ ions in one direction while antiporting H⁺ ions, much in the same way as some natural channels do. E, The future of switch design will necessitate the ability to widely sample geometry space, define energy landscapes, and explicitly incorporate kinetic considerations. The recent advances highlighted here (left: DANCER [34], right: LUCS [36]) have brought us closer to these goals, but a greater number of methods with broader applicability are required to make advanced protein switch design routine.