Comparing the folding landscapes of evolutionarily divergent procaspase-3

All caspases evolved from a common ancestor and subsequently developed into two general classes, inflammatory or apoptotic caspases. The caspase-hemoglobinase fold has been conserved throughout nearly one billion years of evolution and is utilized for both the monomeric and dimeric subfamilies of apoptotic caspases, called initiator and effector caspases, respectively. We compared the folding and assembly of procaspase-3b from zebrafish to that of human effector procaspases in order to examine the conservation of the folding landscape. Urea-induced equilibrium folding/unfolding of procaspase-3b showed a minimum three-state folding pathway, where the native dimer isomerizes to a partially folded dimeric intermediate, which then unfolds. A partially folded monomeric intermediate observed in the folding landscape of human procaspase-3 is not well-populated in zebrafish procaspase-3b. By comparing effector caspases from different species, we show that the effector procaspase dimer undergoes a pH-dependent conformational change, and that the conformational species in the folding landscape exhibit similar free energies. Together, the data show that the landscape for the caspase-hemoglobinase fold is conserved, yet it provides flexibility for species-specific stabilization or destabilization of folding intermediates resulting in changes in stability. The common pH-dependent conformational change in the native dimer, which yields an enzymatically inactive species, may provide an additional, albeit reversible, mechanism for controlling caspase activity in the cell.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A Topological Selection of Folding Pathways from Native States of Knotted Proteins

Understanding how knotted proteins fold is a challenging problem in biology. Researchers have proposed several models for their folding pathways, based on theory, simulations and experiments. The geometry of proteins with the same knot type can vary substantially and recent simulations reveal different folding behaviour for deeply and shallow knotted proteins. We analyse proteins forming open-ended trefoil knots by introducing a topologically inspired statistical metric that measures their entanglement. By looking directly at the geometry and topology of their native states, we are able to probe different folding pathways for such proteins. In particular, the folding pathway of shallow knotted carbonic anhydrases involves the creation of a double-looped structure, contrary to what has been observed for other knotted trefoil proteins. We validate this with Molecular Dynamics simulations. By leveraging the geometry and local symmetries of knotted proteins’ native states, we provide the first numerical evidence of a double-loop folding mechanism in trefoil proteins.

The Promise of Mutation Resistant Drugs for SARS-CoV-2 that Interdict in the Folding of the Spike Protein Receptor Binding Domain

In recent work, we proposed that effective therapeutic drugs aimed at treating the SARS-CoV-2 infection could be developed based on interdicting in the early steps of the folding pathway of key viral proteins, including the receptor binding domain (RBD) of the spike protein. In order to provide for a drug target on the protein, the earliest contact-formation event along the dominant folding pathway of the RBD spike protein was predicted employing the Sequential Collapse Model (SCM). The segments involved in the predicted earliest contact were suggested to provide optimal folding interdiction target regions (FITRs) for potential therapeutic drugs, with a focus on folding interdicting peptides (FIPs). In this paper, we extend our analysis to include 13 known single mutations of the RBD spike protein as well as the triple mutation B1.351 and the recent double mutation B1.617.2. The results show that the location of the FITR does not change in any of the 15 studied mutations, providing for a mutation-resistant drug design strategy for the RBD-spike protein.

Identification of Two Early Folding Stage Prion Non-Local Contacts Suggested to Serve as Key Steps in Directing the Final Fold to Be Either Native or Pathogenic

The initial steps of the folding pathway of the C-terminal domain of the murine prion protein mPrP(90–231) are predicted based on the sequential collapse model (SCM). A non-local dominant contact is found to form between the connecting region between helix 1 and b-sheet 1 and the C-terminal region of helix 3. This non-local contact nucleates the most populated molten globule-like intermediate along the folding pathway. A less stable early non-local contact between segments 120–124 and 179–183, located in the middle of helix 2, promotes the formation of a less populated molten globule-like intermediate. The formation of the dominant non-local contact constitutes an example of the postulated Nature’s Shortcut to the prion protein collapse into the native structure. The possible role of the less populated molten globule-like intermediate is explored as the potential initiation point for the folding for three pathogenic mutants (T182A, I214V, and Q211P in mouse prion numbering) of the prion protein.

Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CH groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CH, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CH when compared to amide groups.

A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding.

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.