Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease.

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of Mpro (MproC) locks Mpro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric Mpro in the early-stage viral replication of both viruses.

The Sensitivity of Computational Protein Folding to Contact Map Perturbations: The Case of Ubiquitin Folding and Function.

Ubiquitin is a small model protein, commonly used in protein folding experiments and simulations. We simulated ubiquitin using a well-tested structure-based model coarse-grained to a Cα level (Cα-SBM) and found that the simulated folding route did not agree with the experimentally observed one. Simulating the Cα-SBM with a cutoff contact map, instead of a screened contact map, switched the folding route with the new route matching the experimental route. Thus, the simulated folding of ubiquitin is sensitive to contact map definition. The screened contact map, which is used in folding simulations because it captures protein folding cooperativity, removes contacts in which the atoms in contact are occluded by a third atom and is less sensitive to the value of the cutoff distance in well-packed regions of the protein. In sparsely packed regions, the larger cutoff distance creates bridging contacts between atoms which are separated by voids. Such contacts do not seem to affect the folding of most proteins, including those of the ubiquitin fold. However, the surface of ubiquitin has several protruding functional side chains which naturally create bridging contacts. Together, our results show that subtle structural features of a protein that may not be apparent by mere observation can be identified by comparing folding simulations of SBMs in which these features are differently encoded. When such structural features are preserved for functional reasons, differences in computational folding can be leveraged to identify functional features. Notably, such features are accessible to a gradation of SBMs even in commonly studied proteins such as ubiquitin.

Intrinsic Disorder in a Well-Folded Globular Protein.

The folded structure of the heterodimeric sweet protein monellin mimics single-chain proteins with topology ß1-α1-ß2-ß3-ß4-ß5 (chain A: ß3-ß4-ß5; chain B: ß1-α1-ß2). Furthermore, like naturally occurring single-chain proteins of a similar size, monellin folds cooperatively with no detectable intermediates. However, the two monellin chains, A and B, are marginally structured in isolation and fold only upon binding to each other. Thus, monellin presents a unique opportunity to understand the design of intrinsically disordered proteins that fold upon binding. Here, we study the folding of a single-chain variant of monellin (scMn) using simulations of an all heavy-atom structure-based model. These simulations can explain mechanistic details derived from scMn experiments performed using several different structural probes. scMn folds cooperatively in our structure-based simulations, as is also seen in experiments. We find that structure formation near the transition-state ensemble of scMn is not uniformly distributed but is localized to a hairpin-like structure which contains one strand from each chain (ß2, ß3). Thus, the sequence and the underlying energetics of heterodimeric monellin promote the early formation of the interchain interface (ß2-ß3). By studying computational scMn mutants whose "interchain" interactions are deleted, we infer that this energy distribution allows the two protein chains to remain largely disordered when this interface is not folded. From these results, we suggest that cutting the protein backbone of a globular protein between residues which lie within its folding nucleus may be one way to construct two disordered fragments which fold upon binding.