Biology subjects

Augustyniak, R.

Publications and source records attributed to Augustyniak, R..

4 recordsLinked to original sources

bioRxiv2026

Phosphoserine clusters as metal ion sensors for protein phase separation

Phosphorylation is a major regulator of biomolecular condensation, yet it remains unclear whether clustered phosphoserines can directly tune phase behavior via metal-ion coordination. Here, using solution NMR spectroscopy and human heterochromatin protein 1 (HP1) as a model system, we show that stepwise phosphorylation of its N-terminal serine cluster generates a dynamic metal-responsive module that engages Mg{superscript 2}, Ca{superscript 2}, and Mn{superscript 2}, whereas the unmodified protein shows little or no response. Metal coordination lowers the saturation concentration of phosphorylated HP1, reshapes the temperature-dependent stability of its condensates, and modulates the effects of peptide regulators in an ion-specific manner. Our data support a model in which weak, transient metal-mediated contacts enhance intermolecular connectivity between phosphorylated HP1 molecules, promoting reversible condensation alongside canonical electrostatic interactions. These findings establish clustered phosphoserines as sequence-encoded metal-responsive elements that couple post-translational modification to the material properties of biomolecular condensates.

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↗

bioRxiv2017

Structure of a AAA+ unfoldase in the process of unfolding substrate

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 [A] stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

biochemistry↗