Biology subjects

Bruno da Silva, F.

Publications and source records attributed to Bruno da Silva, F..

3 recordsLinked to original sources

bioRxiv2025

Multi-barrier unfolding of the double-knotted protein, TrmD-Tm1570, revealed by single-molecule force spectroscopy and molecular dynamics

The doubly knotted motif is one of the least expected features in proteins, occurring in both globular and transmembrane forms. Here, we focus on globular protein members of the methyltransferase family: the TrmD-Tm1570 protein, which contains two deep 31 knots, and the single-knotted proteins TrmD and Tm1570, all from Calditerrivibrio nitroreducens. Using various biophysical experimental techniques and computer simulations with AI-based methods, we studied their thermal and thermodynamic stability, as well as their mechanical unfolding. Based on molecular dynamics (MD) simulations, with the Structure-Based C Model (SBM-C) and UNRES (coarse-grained), we show that native contacts alone are not sufficient to fold double-knotted proteins. However, native contacts are sufficient to fold the single-knotted proteins TrmD and Tm1570 into their native conformations. Using the same model, we identified four possible unfolding and untying pathways, in which each domain can self-tie independently at some stage of the process. Optical tweezers (OT) experiments show that this process is also reversible, although the stretched state remains knotted. In addition, we observed higher thermal and mechanical stability in Tm1570 compared with TrmD, which is partly attributable to the position of the knot core. Overall, our results suggest that double-knotted protein from the SPOUT family can only partially self-fold, and that full knotting may require the assistance of a chaperone.

biophysics↗

bioRxiv2025

Universe of Lasso Proteins: Exploring the limit of entanglement and folding landscape of proteins predicted by AlphaFold

AO_SCPLOWBSTRACTC_SCPLOWKnots and lasso topology represent a class of natural motifs found in proteins which are characterized by a threaded structure. Proteins with a lasso motif represent a macroscopic version of the peptide lasso, which are known for their high stability and offer tremendous potential for the development of novel therapeutics. Here, based on AlphaFold, we have shown the limit of topological complexity of naturally occurring protein structures with cysteine bridges. Based on 176 million high confidence (pLDDT > 70) AlphaFold-predicted protein models and a detailed analysis of the conservation of the motif in a family, we found four new lasso motifs, including L4 and LS4LS3 topologies, and the first examples of knotted lasso proteins: L1K31 and L3#K31. We show that in the case of natural proteins, there are no lassos with 5 threadings but there exist some with 6. Families possessing proteins with more than 6 threadings did not exceed the conservation threshold of 10%. Moreover, we propose a probable folding mechanism for the LS4LS3 lasso motif, enhancing our view on protein folding and stability. This work expands the topological space of lasso type motifs in proteins but also suggests that more complex structures could be unfavorable for proteins. HighlightsO_LIDiscovery of novel non-trivial lasso motifs: the L4, supercoiling of both tails LS4LS3, and the first knotted lasso proteins: L1K31 and L3#K31. C_LIO_LIThe knotted lasso motifs are in membrane proteins. C_LIO_LILassos topologies with 5 or more crossings are not conserved in protein families, and more complex motifs do not exist C_LIO_LI472 new InterPro entries with a high probability of non-trivial lasso motif C_LIO_LIPotential folding pathway for proteins with complex supercoiled lasso motif LS4LS3 C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/644650v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@9c3cedorg.highwire.dtl.DTLVardef@163f941org.highwire.dtl.DTLVardef@811e31org.highwire.dtl.DTLVardef@e53de5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

First crystal structure of double knotted protein TrmD-Tm1570 - inside from degradation perspective

Herein, we present the first crystal structure of a double knotted protein TrmD-Tm1570 from Calditerrivibrio nitroreducens, as well the X-ray structure of each sub-domain. The protein consists of two domains TrmD and Tm1570, each embedding a single trefoil knot, which can function on their own. TrmD-Tm1570 forms a compact homodimeric complex. This protein represents one of 296 possible doubly knotted proteins from SPOUT family. Based on TrmD-Tm1570 from Calditerrivibrio nitroreducens we show that a double knotted protein can be fully degraded by the ClpXP degradation system, as well as its individual domains. We used numerical simulations to explain the difference in the speed of degradation. The derived kinetic parameters for the degradation process are comparable to the experimental data found for unknotted polypeptide chains.

biophysics↗