Biology subjects

Faisca, P. F.

Publications and source records attributed to Faisca, P. F..

6 recordsLinked to original sources

bioRxiv2026

The Gordian Knot Enhances Ubiquitin Binding in UCH-L1

UCH-L1 is a monomeric deubiquitinating enzyme whose native structure embeds a shallow $5_2$ knot located near the N-terminus, placing the knotted topology in direct proximity to both the substrate-binding pocket and the catalytic site. While our previous work established that N-terminal integrity is critical for catalytic activity, the energetic cost of unknotting and its structural consequences remained unquantified. Here, we combine steered molecular dynamics with an umbrella sampling scheme to generate topologically modified variants of UCH-L1 and, for the first time, reconstruct the free-energy profile of UCH-L1 unknotting. The potential of mean force reveals a steep energetic barrier to knot disruption, consistent with knotting being a late, rate-limiting folding step that is effectively locked in once the native structure is established. Long unbiased MD simulations of fully unknotted variants in both apo and holo states show that knot removal increases local flexibility at the N-terminus without inducing significant global structural destabilization. Binding energy calculations indicate that the unknotted variant binds to ubiquitin less tightly than the wild-type ($\sim$-62~vs~$\sim$-76~kcal/mol), suggesting that topological integrity contributes to substrate affinity. Together, these results show that the $5_2$ knot in UCH-L1 is not a passive structural feature but a functional element that fine-tunes folding kinetics and contributes to substrate binding efficiency.

biophysics↗

bioRxiv2025

Folding and knotting of biotic and pre-biotic amino acid sequences through reverse evolution

We develped a simple reverse evolution method to explore the protein folding transition and knotting process in globular proteins throughout evolution using as a proxy for evolutionary time the lenght of the amino acid alphabet. Three small proteins were considered. An unknotted one featuring a beta-sandwhich fold (FN3), a protein embedding a shallow trefoil knot (MJ0366), and a deeply knotted trefoil protein (YibK). Results from Monte Carlo simulations of a native-centric C model show that thermal stability increases througout evolution for all considered proteins suggesting it provided a selective pressure for the introduction of biosynthetic amino acids in the alphabet repertoire. Additionally, the thermodynamic cooperativity, the two-state nature of the folding transition, and the kinetic efficiency of folding (and knotting) displayed by the unknotted (and shallow knotted) protein have been roughly conserved throughout evolution, indicating that early alphabets, and, in particular the anscestral one composed of 10 amino acids, is competent to self-assemble relatively complex native structures. However, while the ancestral alphabet is clearly more efficient in collapsing the polypetpide chain to non-specific (i.e. non-native) knots, it significantly compromises the thermodynamic cooperativity and the folding ability of the deeply knotted protein YibK. The findings presented here collectively suggest that the alphabet of amino acids evolved to improve folding, but early alphabets might not have favoured the folding of native structures with deep knots. This could help to explain why deep knots in proteins are statistically uncommon.

biophysics↗

bioRxiv2023

Effects of sequence-dependent non-native interactions in equilibrium and kinetic folding properties of knotted proteins

Determining the role of non-native interactions in folding dynamics, kinetics and mechanisms is a classical problem in protein folding. More recently, this question has witnessed a renewed interest in light of the hypothesis that knotted proteins require the assistance of non-native interactions to fold efficiently. Here, we conducted extensive equilibrium and kinetic Monte Carlo simulations of a simple off-lattice C-alpha model to explore the role of non-native interactions in the thermodynamics and kinetics of three proteins embedding a trefoil knot in their native structure. We find that equilibrium knotted conformations are stabilized by non-native interactions that are non-local, and proximal to native ones, thus enhancing them. Additionally, non-native interactions increase the knotting frequency at high temperature, and in partially folded conformations below the transition temperature. While non-native interactions clearly enhance the efficiency of the transition from an unfolded conformation to a partially folded knotted one, they are not required to efficiently fold a knotted protein. Indeed, a native centric interaction potential drives the most efficient folding transition, provided that the simulation temperature is well below the transition temperature of the considered model system.

biophysics↗

bioRxiv2021

Predicting stable binding modes from simulated dimers of the D76N mutant of β2-microglobulin

The D76N mutant of the {beta}2m protein is a biologically motivated model system to study protein aggregation. There is strong experimental evidence, supported by molecular simulations, that D76N populates a highly dynamic conformation (which we originally named I2) that exposes aggregation-prone patches as a result of the detachment of the two terminal regions. Here, we use Molecular Dynamics simulations to study the stability of an ensemble of dimers of I2 generated via protein-protein docking. MM-PBSA calculations indicate that within the ensemble of investigated dimers the major contribution to interface stabilization at physiological pH comes from hydrophobic interactions between apolar residues. Our structural analysis also reveals that the interfacial region associated with the most stable binding modes are particularly rich in residues pertaining to both the N- and C-terminus, as well residues from the BC- and DE-loops. On the other hand, the less stable interfaces are stabilized by intermolecular interactions involving residues from the CD- and EF-loops. By focusing on the most stable binding modes, we used a simple geometric rule to propagate the corresponding dimer interfaces. We found that, in the absence of any kind of structural rearrangement occurring at an early stage of the oligomerization pathway, some interfaces drive a self-limited growth process, while others can be propagated indefinitely allowing the formation of long, polymerized chains. In particular, the interfacial region of the most stable binding mode reported here falls in the class of self-limited growth. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LIThe D76N mutant of protein {beta}2m populates an aggregation-prone monomer (I2) with unstructured terminal regions C_LIO_LIMolecular Dynamics simulations and MM-PBSA calculations indicate that dimers of I2 are stabilized by hydrophobic interactions C_LIO_LIThe N- and C-terminal regions, together with the BC- and DE-loops are prevalent in the most stable dimer interfaces, while the CD- and EF-loop appear in the less stable ones C_LIO_LIThe most stable dimer interface has a limited potential to oligomerize in the absence of structural rearrangement C_LI

biophysics↗

bioRxiv2021

A specific set of heterogeneous native interactions yields efficient knotting in protein folding

Native interactions are crucial for folding, and non-native interactions appear to be critical for efficiently knotting proteins. Therefore, it is important to understand both their roles in the folding of knotted proteins. It has been proposed that non-native interactions drive the correct order of contact formation, which is essential to avoid backtracking and efficiently self-tie. In this study we ask if non-native interactions are strictly necessary to tangle a protein, or if the correct order of contact formation can be assured by a specific set of native, but otherwise heterogeneous, interactions. In order to address this problem we conducted extensive Monte Carlo simulations of lattice models of proteinlike sequences designed to fold into a pre-selected knotted conformation embedding a trefoil knot. We were able to identify a specific set of heterogeneous native interactions that drives efficient knotting, and is able to fold the protein when combined with the remaining native interactions modeled as homogeneous. This specific set of heterogeneous native interactions is strictly enough to efficiently self-tie. A distinctive feature of these native interactions is that they do not backtrack, because their energies ensure the correct order of contact formation. Furthermore, they stabilize a knotted intermediate state, which is enroute to the native structure. Our results thus show that - at least in the context of the adopted model - non-native interactions are not necessary to knot a protein. However, when they are taken into account into protein energetics it is possible to find specific, non-local non-native interactions that operate as a scaffold that assists the knotting step.

biophysics↗

bioRxiv2021

The folding space of protein β2-microglobulin is modulated by a single disulfide bond

Protein beta-2-microglobulin ({beta}2m) is classically considered the causative agent of dialysis related amyloidosis (DRA), a conformational disorder that affects patients undergoing long-term hemodialysis. Together with the wild type form, the {Delta}N6 structural variant, and the D76N mutant, have been extensively used as model systems of {beta}2m aggregation. In all of them, the native structure is stabilized by a disulfide bridge between the sulphur atoms of the cysteine residues 25 (at B strand) and 80 (at F strand), which has been considered fundamental in {beta}2m fibrillogenesis. Here, we use extensive Discrete Molecular Dynamics simulations of a full atomistic structurebased model to explore the role of this disulfide bridge as a modulator of the folding space of {beta}2m. In particular, by considering different models for the disulfide bridge, we explore the thermodynamics of the folding transition, and the formation of intermediate states that may have the potential to trigger the aggregation cascade. Our results show that the dissulfide bridge affects folding transition and folding thermodynamics of the considered model systems, although to different extents. In particular, when the interaction between the sulphur atoms is stabilized relative to the other intramolecular interactions, or even locked (i.e. permanently established), the WT form populates an intermediate state featuring a well preserved core, and two unstructured termini, which was previously detected only for the D76N mutant. The formation of this intermediate state may have important implications in our understanding of {beta}2m fibrillogenesis.

biophysics↗