Biology subjects

Walczewska-Szewc, K.

Publications and source records attributed to Walczewska-Szewc, K..

6 recordsLinked to original sources

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

biochemistry↗

bioRxiv2026

Dynamic single-molecule binding of carbamazepine explains diffuse cryo-EM density in the SUR1 drug-binding pocket

Carbamazepine (CBZ), an anticonvulsant drug and pharmacochaperone of pancreatic ATP-sensitive potassium (KATP) channels, binds within the SUR1 drug-binding pocket. However, cryo-electron microscopy (cryo-EM) studies revealed an unusually diffuse ligand-associated density that could not be unambiguously interpreted. Despite being substantially smaller than glibenclamide (GBM), CBZ produced a density of comparable size, leading to the proposal that either two CBZ molecules occupy the binding pocket simultaneously or that a single molecule adopts multiple binding configuresions. Here, we combine molecular dynamics simulations, density reconstruction from simulation ensembles, quantum chemical calculations, and binding free-energy analyses to investigate the molecular origin of this diffuse cryo-EM density. Density functional theory calculations indicate that CBZ dimers are only weakly stabilized and do not exhibit a strong intrinsic preference for dimerization. Molecular dynamics simulations further show that two simultaneously bound CBZ molecules fail to form a stable dimeric complex and generate density distributions inconsistent with the experimental cryo-EM map. In contrast, a single CBZ molecule remains localized within the SUR1 cavity while sampling a broad ensemble of positions and orientations. Remarkably, density reconstructed directly from the simulation trajectories closely reproduces the extent and shape of the experimentally observed cryo-EM density. Our results demonstrate that the diffuse cryo-EM density associated with CBZ arises from dynamic binding of a single molecule rather than ligand dimerization. More broadly, this study highlights how ligand dynamics can shape cryo-EM densities and illustrates the value of integrating molecular simulations with experimental structural data to interpret heterogeneous ligand-binding states.

Molecular Biology↗

bioRxiv2025

Evaluating the ligands' potency to modulate the fast inactivation of voltage-gated sodium channel.

Electrical impulse transmission along the nerve fiber in the form of the action potential is possible due to fast conformational changes of voltage-gated sodium channels (Nav) that control the sodium ions flow into the cell. The transition between functional states, called the gating mechanism, can be modulated by natural toxins and drugs. Here, we propose to use steered molecular dynamics (SMD) to investigate the ability of various ligands to impact the gating of P. americana cockroach Nav. By calculating mechanical forces required to relocate the inactivation particle to its binding pocket or to dislocate it, we assessed ligands efficacy in trapping a channel in a given state (open or fast inactivated). Importantly, we showed that sulfonamide PF-05089771 and phospholipid PIP2 act as insect Nav channels inhibitors. We confirmed the ligands action by electrophysiological measurements of their ability to modulate the neural activity. Our approach, applied here on a cockroach channel, can be used in any other Nav, i.g, to evaluate new drug candidates.

biophysics↗

bioRxiv2025

multiSMD - a Python toolset for multidirectional Steered Molecular Dynamics

Understanding the direction-dependence of molecular interactions is critical for elucidating biological processes such as protein-protein binding, ligand dissociation, and mechanotransduction. While steered molecular dynamics (SMD) simulations enable the study of force-induced transitions, conventional single-direction approaches may overlook anisotropic responses inherent to biomolecular systems. Here, we present multiSMD, a Python-based tool that automates the setup and analysis of multi-directional SMD simulations in NAMD and GROMACS. By systematically probing forces along multiple spatial vectors, multiSMD captures direction-dependent phenomena--such as varying energy barriers or structural resilience--that remain hidden in traditional SMD. We demonstrate the utility of our approach through three distinct applications: (i) anisotropic unbinding in a protein-protein interaction, (ii) ligand dissociation pathways dependent on pulling direction, and (iii) force-induced remodeling of intrinsically disordered regions. multiSMD streamlines the exploration of mechanical anisotropy in biomolecules, offering a computational framework to guide experiments (e.g., AFM or optical tweezers) and uncover mechanistic insights inaccessible to single-axis methods. Availability and implementation: multiSMD is freely available at https://github.com/kszewc/multiSMD

bioinformatics↗

bioRxiv2024

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone

Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet(R) followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) [~] 9 M. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

biochemistry↗

bioRxiv2022

Structural insights into ATP-sensitive potassium channel mechanics: a role of intrinsically disordered regions.

Commonly used techniques, such as CryoEM or Xray, are not able to capture the structural reorganizations of disordered regions of proteins (IDR), therefore it is difficult to assess their functions in proteins based exclusively on experiments. To fill this gap, we used computational molecular dynamics simulations methods to capture IDR dynamics and trace biological function-related interactions in the Kir6.2/SUR1 potassium channel. This ATP-sensitive octameric complex, one of the critical elements in the insulin secretion process in human pancreatic {beta}-cells, has four to five large, disordered fragments. Using unique MD simulations of the full Kir6.2/SUR1 channel complex, we present an in-depth analysis of the dynamics of the disordered regions and discuss the possible functions they could have in this system. Our MD results confirmed the crucial role of the N-terminus of the Kir6.2 fragment and the L0-loop of the SUR1 protein in the transfer of mechanical signals between domains that trigger insulin release. Moreover, we show that the presence of IDRs affects natural ligands binding. Our research takes us one step further towards understanding the action of this vital complex.

molecular biology↗