Design of stimulus-responsive two-state hinge proteins

Abstract

In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled.

Computational Design of Periplasmic Binding Protein Biosensors Guided by Molecular Dynamics

“…A wider variety of candidate insertion sites will help with the successful insertions of a wider variety of effectors such as transcription factors and enzymes (Guntas and Ostermeier, 2004; Younger et al ., 2018). It would also be interesting to see this method applied to other sensor proteins with similar hinge-like binding dynamics, such as the de novo hinge proteins recently developed in the Baker lab (Praetorius et al ., 2023). Finally, the most immediate impact of this work is the forging of a new, computational avenue for PBP-biosensor discovery that can be performed at a much greater scale than current wet-lab methods for minimal labour and cost.…”

De novo design of allosterically switchable protein assemblies

“…We began by designing proteins that can adopt two different oligomeric ring states that differ in their radius and number of subunits ( Fig 1b ). Within the monomeric subunits of these oligomers, we embed a two-state “hinge” module 27 that can toggle between two structurally defined alternative conformations, a closed “X” state, and an open “Y” state, the latter of which presents a groove that can bind to an effector peptide with nanomolar affinity 11 . In the absence of the effector, the “X” predominates, while the “Y” dominates in the presence of saturating amounts of effector ( Fig 1c , EFig.1a, ΔG1 in Fig 1b ).…”

“…To determine if the two-state, conformationally switchable nature of the ring is essential for binding cooperativity, we generated a static version of sr312 (sr312_locked) containing two cysteine mutations in the hinge region which, under oxidizing conditions, form a disulfide bridge that locks the hinge into the Y state 11 and thus locks the ring in the Y4 state ( Fig 4g ) . nsEM on this construct shows that, after oxidation, the protein assembles into C4-symmetric tetramers comparable to the peptide-bound Y4 state of sr312 even without peptide ( Fig 4g , EFig.20g ).…”

“…However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge 8,9 . Here, inspired by the classic Monod-Changeux-Wyman model of cooperativity 10 , we investigate the de novo design of allostery through rigid-body coupling of designed effector-switchable hinge modules 11 to protein interfaces 12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to effector binding and dihedral cages that undergo effector-induced disassembly.…”

Mutational destabilisation accelerates the evolution of novel sensory and network functions

“…On the contrary, an over-reliance on allosteric regulators could lead to a reduction in the evolvability of metabolic networks. New allosteric regulators can only be created when a specific binding interface and a mechanism to transmit the new interaction into a clear structural transition have emerged simultaneously 11,12 . This is in addition to the constraint that metabolic regulators fulfil an original function within the existing network.…”