Biology subjects

Machuqueiro, M.

Publications and source records attributed to Machuqueiro, M..

4 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↗

bioRxiv2022

Interfacial dynamics and growth modes of β2-microglobulin dimers

Protein aggregation is a complex process that strongly depends on environmental conditions and has considerable structural heterogeneity, not only at the level of fibril structure but also at the level of molecular oligomerization. Since the first step in aggregation is the formation of a dimer, it is important to clarify how certain properties (e.g., stability or the interface geometry) of the latter may determine the outcome of aggregation. Here, we developed a simple model that represents the dimers interfacial region by two angles (spanning the so-called growth landscape), and investigate how modulations of the interfacial region occurring on the ns-s timescale change the dimers growth mode. We applied this methodology to 15 different dimer configurations of the {beta}2m D76N mutant protein equilibrated with long MD simulations and identified which of them have limited and unlimited growth modes, with different consequences to their aggregation potential. We found that despite the highly dynamic nature of the starting configurations, most polymeric growth modes tend to be conserved within the studied time scale. The proposed methodology performs remarkably well taking into consideration that the {beta}2m dimers are formed by monomers with detached termini, and their interfaces are stabilized by non-specific apolar interactions, leading to relatively weak binding affinities.

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↗

bioRxiv2019

Dynamical rearrangement of human epidermal growth factor receptor 2 upon antibody binding: effects on the dimerization

Human epidermal growth factor 2 (HER2) is a ligand-free tyrosine kinase receptor of the HER family that is overexpressed in some of the most aggressive tumours. Treatment of HER2+ breast cancers with the humanized monoclonal anti-HER2 antibody (Trastuzumab) revealed highly effective, encouraging the development of various HER2-specific antibodies, kinase inhibitors and dimerization inhibitors for cancer therapy. Although it is known that HER2 dimerization involves a specific region of its extracellular domain, the so-called \"dimerization arm\", the mechanism of dimerization inhibition remains uncertain. However, uncovering how antibody interactions lead to inhibition of HER2 dimerization is of key importance in understanding its role in tumour progression and therapy. Herein, we employed several computational modelling techniques for a molecular-level understanding of the interactions between HER and specific anti-HER2 antibodies, namely an antigen-binding (Fab) fragment (F0178) and a single chain variable fragment from Trastuzumab (scFv). Specifically, we investigated the effects of antibody-HER2 interactions on the key residues of \"dimerization arm\" from molecular dynamics (MD) simulations of unbound HER (in a total of 1 {micro}s), as well as scFv:HER2 and F0178:HER2 complexes (for a total of 2.5 {micro}s). A deep surface analysis of HER receptor revealed that the binding of specific anti-HER2 antibodies induced conformational changes both in the interfacial residues, which was expected, and in the ECDII, in particular at the \"dimerization arm\", which is critical in establishing protein-protein interface (PPI) interactions. Our results support and advance the knowledge on the already described trastuzumab effect on blocking HER2 dimerization through synergistic inhibition and/or steric hindrance. Furthermore, our approach offers a new strategy for fine-tuning target activity through allosteric ligands.\n\nAuthor summaryIncreasing insight into the genetics and molecular biology of diseases has resulted in the identification of a high number of potential molecular targets for drug discovery and development. Human Epidermal Growth Factor Receptor 2 (HER2) is one of the most relevant Epidermal Growth Factor Receptor (EGFR) members, whose overexpression has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. Thus, the development of novel approaches on anti-HER2 therapies is quiet relevant. Molecular modelling and simulation can be used to bring new perspectives, both in structure-based drug design and to provide atomistic information of the intermolecular coupling dynamics between inhibitors and receptors via interactive computographic software. Considering my interest in drug design and mechanisms of action using integrated in silico approaches, herein, I have used multiple methods to evaluate how HER2 coupling to its different partners could alter this functional mechanism. The results suggest that the antibodies fragments studied show different dynamic complexes with HER2 although both could contribute to downstream of the tumour cell receptor pathways. I do believe that future research breakthroughs with aid of chemo-bioinformatics will allow a more comprehensive perception on biomedicine.

bioinformatics↗