Biology subjects

Vreede, J.

Publications and source records attributed to Vreede, J..

8 recordsLinked to original sources

bioRxiv2026

GerAB Residues Predicted to Interact with Water Based on MD Simulations Mediate Germinosome Stability in Bacillus subtilis spores

Some species in the Bacillales and Clostridiales orders form spores under unfavourable environmental conditions. These spores are metabolically dormant and highly resistant to extreme stress. The spore core--analogous to the protoplast of vegetative cells--contains only 25-45% water by wet weight, compared to ~80% in vegetative cells. Upon activation by small-molecule nutrients, spores germinate, restoring their core water content, restoring metabolism and becoming easy to kill, while progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA GR (germinant receptor), a membrane protein belonging to the Amino Acid--Polyamine-Organocation (APC) superfamily of transporters. It functions as the L-alanine sensor that initiates germination and was previously predicted, based on molecular dynamics (MD) simulations, to contain a putative water channel. Using MD simulations, we identified low amount of water permeating through GerAB (ranging from 1-121 water molecule/{micro}s in 10 parallel MD simulations), thus revealing a water pathway in GerAB that diverges from the L-alanine binding pocket, suggesting that water transport may play roles in germination beyond facilitating ligand binding. Analysis of water-residue contact frequencies identified eight hydrophilic residues lining this path. Individual substitution of high-contact residues with similarly sized non-polar residues impaired L-alanine germination and disrupted GerAB structural integrity as assessed by Western Blotting. These mutants also respond to the AGFK germinant mixture (L-asparagine, D-glucose, D-fructose and potassium) in slower, yet individually distinct kinetics compared to that of wt spores. These findings prove that water contact residues in GerAB predicted by MD simulations are crucial for the stability of this protein and thus the germinosome complex with all GRs.

microbiology↗

bioRxiv2025

Nuclear mechanostability emerges from satellite DNA condensation into chromocenters

As the largest organelle, the nucleus endures significant mechanical stresses over the cellular lifespan, and mechanostability, i.e. the ability to resist deformation, is critical for genome integrity and function. Here, we reveal that nuclear mechanostability is an emergent property arising from the clustering of satellite DNA repeats into nuclear condensates known as chromocenters. Targeted chromocenter disruption in Drosophila testes subjected to natural and artificial mechanical stress compromises nuclear mechanostability, leading to deformed nuclei, DNA damage, and chromosome breaks. Conversely, enhancing chromocenter coalescence through genetic means improves mechanostability. Molecular dynamics simulations suggest that chromocenters enable physically linked chromosomes to respond collectively, rather than individually, to mechanical challenge, and dissipate external forces over a larger nuclear surface. We propose that the satellite DNA-dependent mechanostability framework described here likely extends to other cells and tissues facing mechanical stress, and offers an explanation for the evolutionary success of these non-coding repeats across eukaryotes.

cell biology↗

bioRxiv2025

MDNA: a software module for DNA structure generation and analysis

Exploring the dynamical and structural properties of molecular complexes involving DNA is a fundamentally important aspect of understanding many biological processes. Although tools exist for modeling linear DNA and simple complexes, significant challenges remain in generating intricate biomolecular assemblies and incorporating biologically relevant modifications. These limitations restrict the ability to create accurate starting configurations for advanced molecular simulation studies. Here, we introduce MDNA, a molecular modeling toolkit that bridges these gaps by enabling the construction and analysis of complex DNA structures. MDNA offers a versatile solution to generate DNA shapes using a spline-based mapping technique that enables the construction of DNA configurations with arbitrary shapes. Key features include support for (non-)canonical base modifications, such as Watson-Crick-Franklin to Hoogsteen transitions, DNA methylation, and the ability to refine structures using Monte Carlo minimization. The toolkit also provides geometric analysis tools based on rigid body formalism to evaluate DNA structures and trajectories. Together, these features enable users to model and analyze DNA configurations in high detail with a modular Python interface. By integrating structure generation and analysis into a single workflow, MDNA improves the study of DNA-protein interactions, paving the way for new insights into DNA dynamics and molecular simulations.

biophysics↗

bioRxiv2024

Identification of key residues in water channel formation in the Bacillus subtilis germinant receptor protein GerAB

Some Bacillales and Clostridiales bacteria form spores in unfavorable environments. These spores are dormant but can rapidly resume metabolism in response to certain small molecule nutrients. This process is termed germination and can be initiated by a variety of low molecular wt nutrients termed germinants. Structural modeling and mutagenesis studies showed that GerAB, an inner membrane (IM) protein of the Bacillus subtilis spore germinant receptor (GR) GerA, is involved in L-alanine-initiated spore germination. A previous molecular simulation study also suggested there is a water channel in GerAB. In the current work, Steered Molecular Dynamics (SMD) simulations were employed to force a single water molecule through GerAB, identifying three key amino acid residues, Y97, L199 and F342, that interfere with water passage. When these residues were altered to alanine, L-alanine germination was minimal in spores with L199A, F342A and triA (Y97A, L199A and F342A triple mutant), while Y97A mutant spores germinated [~]61%. And besides Y97A, all other mutants showed compromised germination triggered by AGFK mixture. Western blotting found reduced levels of the GerA GR in the Y97A mutant, and an absence of the GerA GR in all other mutants. This proved that all three identified residues are crucial to the structural integrity of the GerAB protein and therefore probably the formation of the GR complex, the germinosome.

microbiology↗

bioRxiv2021

Sequence dependence of transient Hoogsteen base-pairing in DNA

Hoogsteen (HG) base-pairing is characterized by a 180{degrees} rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A{middle dot}T pair in A6-DNA. We observe the known effect of A{middle dot}T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNAs relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.

biophysics↗

bioRxiv2020

Path sampling simulations reveal how the Q61L mutation restricts the dynamics of KRas

The GTPase KRas is a signaling protein in networks for cell differentiation, growth, and division. KRas mutations can prolong activation of these networks, resulting in tumor formation. When active, KRas tightly binds GTP. Several oncogenic mutations affect the conversion between this rigid state and inactive, more flexible states. Detailed understanding of these transitions may provide valuable insights into how mutations affect KRas. Path sampling simulations, which focus on transitions, show KRas visiting several states, which are the same for wild type and the oncogenic mutant Q61L. Large differences occur when converting between these states, indicating the dramatic effect of the Q61L mutation on KRas dynamics. For Q61L a route to the flexible state is inaccessible, thus shifting the equilibrium to more rigid states. Our methodology presents a novel way to predict dynamical effects of KRas mutations, which may aid in identifying potential therapeutic targets. Author summaryCancer cells frequently contain mutations in the protein KRas. However, KRas is a challenging target for anti-cancer drugs, in part because the dynamic behavior of flexible regions in the protein is difficult to characterize experimentally, and occurs on timescales that are too long for straightforward molecular dynamics simulations. We have used path sampling, an advanced simulation technique that overcomes long timescales, to obtain atomistic insight into the dynamics of KRas. Comparing the oncogenic mutant Q61L to the wild type revealed that the mutation closes off one transition channel for deactivating KRas. Our approach opens up the way for predicting the dynamical effects of mutations in KRas, which may aid in identifying potential therapeutic targets.

cancer biology↗

bioRxiv2019

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.\n\nHere, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

biochemistry↗

bioRxiv2016

Environment driven conformational changes modulate H-NS DNA bridging activity

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria, and is believed to be a crucial player in bacterial chromatin organization via its DNA bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the ability of H-NS to bridge DNA. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a \"closed\" and an \"open\", bridging competent, conformation driven by environmental cues and interaction partners.

biochemistry↗