Biology subjects

Lewandowska, I.

Publications and source records attributed to Lewandowska, I..

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↗

bioRxiv2023

Nucleolar Essential Protein 1 (Nep1): Elucidation of Enzymatic Catalysis Mechanism by Combined Molecular Dynamics Simulation and Quantum Chemical Calculations

Nep1 is a protein essential for the formation of the eukaryotic and archaeal small ribosomal subunit. It is an enzyme responsible for the site-specific SAM-dependent methylation of pseudouridine ({Psi}) during the pre-rRNA processing. It possesses a non-trivial topology, namely, a 31 knot in the active site. Herein, we investigate the structure and mechanism of catalysis of Nep1 using a combination of bioinformatics, computational, and experimental methods. In particular, we address the issue of seemingly unfeasible deprotonation of {Psi} nucleobase in the active site of Nep1 by a distant aspartate residue (e.g., D101 in Nep1 of S. cerevisiae). Sequence alignment analysis across different organisms identifies a conserved serine/threonine residue that may play a role of a proton-transfer mediator (e.g., S233 in Nep1 from S. cerevisiae), facilitating the reaction. Two enzyme-substrate complexes, one based on an available crystal structure and the other generated by molecular docking, of representative eukaryotic (from S. cerevisiae) and archaeal (from A. fulgidus) Nep1 homologs are subjected to molecular dynamics (MD) simulations. The resulting trajectories confirm that the hydroxyl-containing amino acid can indeed adopt a position suitable for proton-shuttling, with the OH group located in between the proton donor and acceptor. However, during the MD simulations, a water molecule emerges from arrangements of the active site, which can assume the role of the proton-transfer mediator instead. To discern between these two alternative pathways, we evaluate the possible methylation mechanisms by quantum-chemical calculations based on density functional theory, using the cluster approach. The obtained energy profiles indicate that the most facile course of the reaction for both the yeast and archaeal enzymes is to engage the water molecule. These results are corroborated by agreement of the computed energy barriers with experimentally measured enzyme kinetics. Moreover, mutational studies show that, while aspartate D101 is crucial for the catalytic activity, serine S233 is irrelevant in this context, indirectly supporting the water-mediated proton transfer. Our findings comprehensively elucidate the mode of action of Nep1 and provide implication for understanding the catalytic mechanisms of other enzymes that involve a proton transfer in the active site over extended distances.

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↗