Biology subjects

Hanke, A.

Publications and source records attributed to Hanke, A..

10 recordsLinked to original sources

bioRxiv2026

How specific structural differences of Bcl2 proteins modulate the interaction with BH3 domains and apoptotic function

Intrinsic apoptosis is mainly regulated through a network of conserved interactions between Bcl-2 proteins involving hydrophobic binding grooves and BH3 domains. Despite these conserved interfaces, family members exhibit distinct binding affinities and play opposing roles in apoptosis. While static structural differences partially account for this divergence, it remains unclear how opposing apoptotic function reflects in BH3 helix engagement of individual members. Here, we investigate how a BidBH3 peptide engages with the hydrophobic groove of full-length membrane-anchored Bcl-xL and Bax to identify shared and unique features of binding that may relate to distinct apoptotic functions. Using state-of-the-art enhanced-sampling simulations, we mapped the complete binding and folding landscapes of these critical cell-death regulators in membranes. Our simulations align with experimental measurements in terms of predicted absolute binding affinities, and also capture the dynamic, atomistic details of the conformational changes induced by BH3 helices. Together, these details highlight the structural principles of BH3 in-groove engagement that determine apoptotic function, paving the way towards the modulation of the interactions among the Bcl-2 family members.

biophysics↗

bioRxiv2026

Evidenced by Indigenous and Western Science: An Arctic Nation Building Project Threatens Caribou and Inuit Harvesting Rights

Canadas ambition for mineral security and its responsibilities to protect at-risk species and uphold Indigenous rights clash in the case of the Grays Bay Road and Port (GBRP) in Nunavut, an infrastructure project intended to unlock critical mineral deposits. We compiled Indigenous and Western science through a density analysis of caribou harvesting data near the proposed project site. We identified three consistently used harvesting hotspots, with the most significant hotspot lying directly in the path of the proposed GBRP project. These results indicate that the GBRP project will have significant and unmitigable negative effects on caribou conservation, food security, and Inuit harvesting rights. Prime Minister Carney claims that middle power countries must act consistently in this era of geopolitical rupture; this commitment must transfer to natural resource development reviews so that decision-making may be consistent and rooted in cross-legislation responsibilities and values, including the land claims agreements between Indigenous groups and the Government of Canada.

ecology↗

bioRxiv2026

Functional RNA Interference in Hymenoscyphus fraxineus: Hairpin RNA-Induced Gene Silencing Of A Polyketide Synthase-like Gene

European ash dieback caused by the invasive ascomycete species Hymenoscyphus fraxineus poses the most prominent danger to common ash trees (Fraxinus excelsior) in Europe. The disease is widely distributed in Europe and currently no efficient management strategy is available. Host-induced gene silencing and exogenous dsRNA applications have shown great potential for controlling fungal diseases in crop plants. In this study, we reported in silico evidence for the presence of a functional RNA interference pathway in Hymenoscyphus fraxineus. Moreover, we showed that the transgenic expression of a double stranded RNA (dsRNA) leads to inhibition of translation of its target polyketide synthase-like gene, a fungal endogene. We explored whether the dsRNA could be introduced exogenously and demonstrated that H. fraxineus can take up externally applied dsRNA molecules. This study highlights the RNA interference mechanism in H. fraxineus and suggests exoRNA applications as a promising approach to control European ash dieback.

molecular biology↗

bioRxiv2025

Structural and dynamic basis of indirect apoptosis inhibition by Bcl-xL: a case study with Bid

Intrinsic apoptosis is a form of cell death which is activated, executed and inhibited by the Bcl-2 protein family. The structural basis of the inhibition mechanisms remains elusive. Here, we characterize the ensemble structural model of the inhibitory Bcl-xL/tBid complex at the mitochondrial membrane by probing inter-residue distances and dynamic solvent accessibilities complemented by integrative modelling and molecular dynamics simulations. We show that Bcl-xL and tBid form a heterodimer anchored to the membrane by the C-terminal helix of Bcl-xL. The BH3 domain of tBid is wedged between the exposed hydrophobic groove of Bcl-xL and the membrane headgroups, while tBids C-terminal helices remain dynamically engaged with the bilayer. This dynamic architecture sheds light on the mechanism of indirect inhibition of apoptosis. Significance StatementProgrammed cell death, or apoptosis, is a fundamental process that eliminates damaged cells. However, cancer cells often evade this process by overexpressing anti-apoptotic Bcl-2 family proteins, which neutralise their pro-apoptotic counterparts. The structural details of the molecular mechanisms governing this inhibition have remained elusive and up to now, only partial structures could be obtained with high-resolution methods. In this study, we provide the structural model of the inhibitory complex formed by the anti-apoptotic protein Bcl-xL and the pro-apoptotic protein Bid at the mitochondrial membrane using experimental constraints and molecular dynamics simulations. Our work provides the structural basis of one of the inhibitory mechanisms of cell death, offering a new framework for developing strategies to overcome therapeutic resistance in cancer.

biophysics↗

bioRxiv2025

Probing the effect of PEG-DNA interactions and buffer viscosity on tethered DNA in shear flow

DNA flow-stretching is a widely employed, powerful technique for investigating the mechanisms of DNA-binding proteins involved in compacting and organizing chromosomal DNA. We combine single-molecule DNA flow-stretching experiments with Brownian dynamics simulations to study the effect of the crowding agent polyethylene glycol (PEG) in these experiments. PEG interacts with DNA by an excluded volume effect, resulting in compaction of single, free DNA molecules in PEG solutions. In addition, PEG increases the viscosity of the buffer solution. By stretching surface-tethered bacteriophage lambda DNA in a flow cell and tracking the positions of a quantum dot labeled at the free DNA end using total internal reflection fluorescence (TIRF) microscopy, we find that higher PEG concentrations result in increased end-to-end length of flow-stretched DNA and decreased fluctuations of the free DNA end. To better understand our experimental results, we perform Brownian dynamics simulations of a bead-spring chain model of flow-stretched DNA in a viscous buffer that models the excluded volume effect of PEG by an effective attractive interaction between DNA segments. We find quantitative agreement between our model and the experimental results for suitable PEG-DNA interaction parameters.

biophysics↗

bioRxiv2024

Somatic hypermutation-mediated paratope flexibility improves the cross-reactivity of human malaria antibodies

The protective capacity of antibodies targeting circumsporozoite protein on sporozoites of the malaria parasite Plasmodium falciparum (PfCSP) is linked to high affinity and cross-reactivity with the PfCSP central repeat domain and N-terminal junction. However, the role of somatic hypermutation (SHM) in the development of such antibodies remains unclear. Here we define the contributions of SHM to the high affinity and strong repeat and N-junction cross-reactivity of the potent anti-PfCSP monoclonal antibody (mAb) 4493 and of similar antibodies with shared SHM and affinity maturation trajectories. Molecular dynamics simulations reveal that SHM reduces the flexibility of the unbound mAb 4493 but increases the flexibility of the antigen-bound complex, thereby lowering the entropic cost for antigen binding. Furthermore, we identify an inverse relation between antibody affinity and serum stability, which limits the protective capacity of these antibodies. Our study provides molecular level evidence for the different roles that the SHM process plays in increasing VH3-49+V{kappa}3-20 antibody affinity and cross-reactivity and demonstrates how antibody affinity maturation can negatively impact antibody stability and thereby function.

immunology↗

bioRxiv2022

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD-simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

biophysics↗

bioRxiv2020

A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics

The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the proteins druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

biophysics↗

bioRxiv2019

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of DNA target sites. The strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of the DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. Here we use the kinetics of loop closure involving closely spaced (131-151 bp), directly repeated loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-ofplane HJ distortion. This result unequivocally stands in contrast to the square-planar intermediate geometry determined from crystallographic data for the Cre-loxP system and other int-superfamily recombinases. J measurements carried out with an isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex may result from the temporal averaging of diverse right-handed and achiral structures. Applied to Cre recombinase, and other biological systems, our approach bridges the static pictures provided by crystal structures and the natural dynamics of macromolecules in vivo. This approach thus advances a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

biophysics↗

bioRxiv2018

Kinetic Pathways of Topology Simplification by Type-II Topoisomerases in Knotted Supercoiled DNA

The topological state of covalently closed, double-stranded DNA is defined by the knot type K and the linking-number difference {Delta}Lk relative to unknotted relaxed DNA. DNA topoisomerases are essential enzymes that control the topology of DNA in all cells. In particular, type-II topoisomerases change both K and {Delta}Lk by a duplex-strand-passage mechanism and have been shown to simplify the topology of DNA to levels below thermal equilibrium at the expense of ATP hydrolysis. It remains a puzzle how small enzymes are able to preferentially select strand passages that result in topology simplification in much larger DNA molecules. Using numerical simulations, we consider the non-equilibrium dynamics of transitions between topological states (K, {Delta}Lk) in DNA induced by type-II topoisomerases. For a biological process that delivers DNA molecules in a given topological state (K,{Delta}Lk) at a constant rate we fully characterize the pathways of topology simplification by type-II topoisomerases in terms of stationary probability distributions and probability currents on the network of topological states (K,{Delta}Lk). In particular, we observe that type-II topoisomerase activity is significantly enhanced in DNA molecules that maintain a supercoiled state with constant torsional tension. This is relevant for bacterial cells in which torsional tension is maintained by enzyme-dependent homeostatic mechanisms such as DNA-gyrase activity.

molecular biology↗