Engineering protein assemblies with allosteric control via monomer fold-switching

Abstract

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening

“…S4). It may be hypothesized that, along the thermal denaturations, temperature could trigger a transient dimerization of dimers into tetramers (tetrameric intermediate) at moderate temperatures, a temperature-driven oligomerization process that has been observed in other proteins [ 47 , 48 ], before reaching the unfolding temperature and ending in monomer unfolding/dissociation; work on this matter is in progress.…”

Artificial Organelles: Towards Adding or Restoring Intracellular Activity

“…For example, protein cages can be designed in a scalable [242] or adaptable [243] manner by incorporating split inteins or disulfide interactions, respectively. Alternatively, capsid assembly can be controlled allosterically [244] or via chemical, thermal and redox control over metal coordination [245] . By using and exploring all of these features, our understanding of these strategies grows and the road towards designed artificial organelles is paved.…”

Synergistic stabilization of a double mutant in CI2 from anin-celllibrary screen

“…The structures were carefully examined, adjusted and refined with Coot and Refmac5, respectively (Emsley et al, 2010;Murshudov et al, 2011). To make sure that the structures were not in a domain swapped configuration (Campos et al, 2019), molecular replacement solutions were also sought using the domain swapped structure with PDB accession code 6QIZ. These consistently yielded significantly worse statistics.…”