Biology subjects

Lycksell, M.

Publications and source records attributed to Lycksell, M..

5 recordsLinked to original sources

bioRxiv2025

Calcium stabilization of a flexible N-terminal domain in a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are responsible for the rapid conversion of chemical to electrical signals. In addition to the canonical extracellular and transmembrane domains, some prokaryotic pLGICs contain an N-terminal domain (NTD) of unclear structure and function. In one such case, the calcium-sensitive channel DeCLIC, the NTD appears to accelerate gating; however, its evident flexibility has posed a challenge to model building, and its role in calcium sensitivity is unclear. Here we report cryo-EM structures of DeCLIC to the highest resolutions thus far, in circularized lipid nanodiscs. Application of refinement tools enabled definition of multiple calcium-binding sites in each symmetric subunit. Under calcium-free conditions, both symmetric and asymmetric classes could be reconstructed, implicating a heterogeneous ensemble. Behavior of these structures in molecular dynamics simulations was consistent with calcium stabilization of the NTD, an effect that may be conserved in structurally homologous domains across evolution.

biophysics↗

bioRxiv2025

Structure and dynamics of a multidomain ligand-gated ion channel revealed under acidic conditions

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution, including key targets of biophysical studies and therapeutic development. However, the intrinsically allosteric, polymodal, modular nature of gating in this protein family presents persistent challenges to biophysical characterization. The bacterial channel DeCLIC constitutes a provocative model system for structure, function, and dynamics in this family, including a modulatory N-terminal domain (NTD). Previous closed structures of DeCLIC support a rationale for its inhibition by calcium via a site of conserved relevance in other family members; however, mechanisms of gating in DeCLIC and properties of its open state have remained unclear. Here we integrated structure-function methods including cryogenic electron microscopy (cryo-EM), molecular dynamics simulations and small-angle neutron scattering under acidic conditions to characterize a previously unreported conformation of DeCLIC with a fully hydrated pore. In contrast to previous structures, the low-pH open conformation captured by cryo-EM was stable and permeable in simulations, and consistent with solution-phase behavior as measured by small-angle scattering. We further captured an alternative closed state of the channel, evidently promoted by depletion of modulatory calcium at low pH, exhibiting dynamic rearrangements in the N-terninal domain. The expanded-pore structure evidently corresponds to a functional open state of DeCLIC, while calcium-site and NTD dynamics drive channel closure, providing a detailed template for biophysical characterization of modulatory mechanisms in ligand-gated ion channels and related systems. Significance StatementThe bacterial protein DeCLIC is a provocative representative of the pentameric ligand-gated ion-channel family. Like its eukaryotic relatives, DeCLIC is sensitive to external calcium, and incorporates a possibly disordered modulatory domain. But as for many ion channels, the details of DeCLIC activation have remained unclear, as no stable open state has been characterized. Combining biophysical methods, including cryogenic electron microscopy, molecular dynamics simulations, and small-angle neutron scattering, allows us to capture, validate, and characterize an evidently open state of DeCLIC, as well as a closed state with a disordered modulatory region. Details of these structures, their function, and their dynamics allow us to propose a mechanism for DeCLIC gating, as well as a generalized scheme for the larger protein family.

biophysics↗

bioRxiv2022

Automated simulation-based membrane-protein refinement into cryo-EM data

I.The resolution revolution has increasingly enabled single-particle cryogenic electron microscopy (cryo-EM) reconstructions of previously inaccessible systems, including membrane proteins - a category that constitutes a disproportionate share of drug targets. We present a protocol for using density-guided molecular dynamics simulations to automatically refine atomistic models into membrane-protein cryo-EM maps. Using adaptive-force density-guided simulations as implemented in the GROMACS molecular dynamics package, we show how automated model refinement of a membrane protein is achieved without the need to manually tune the fitting force ad hoc. We also present selection criteria to choose the best fit model which balances stereochemistry and goodness-of-fit. The proposed protocol was used to refine models into a new cryo-EM density of the membrane protein maltoporin, either in a lipid bilayer or detergent micelle, and we found that results do not substantially differ from fitting in solution. Fitted structures satisfied classical model-quality metrics and improved the quality and the model-to-map correlation of the X-ray starting structure. Additionally, the density-guided fitting in combination with generalized orientation-dependent all-atom potential (GOAP) was used to correct the pixel-size estimation of the experimental cryo-EM density map. This work demonstrates the applicability of a straightforward automated approach to fitting membrane-protein cryo-EM densities. Such computational approaches promise to facilitate rapid refinement of proteins under different conditions or with various ligands present, including targets in the highly relevant superfamily of membrane proteins. II. STATEMENT OF SIGNIFICANCECryo-EM is an increasingly critical method of structure determination. As data collection and model generation become more efficient, iteratively fitting an experimental density can still require considerable time and expertise. Membrane proteins are particularly important targets in pharmacology and bioengineering, but can present distinctive challenges to data quality and modeling. Here, we tested a new tool to drive density fitting with molecular dynamics simulations, in context of a new structure of the membrane protein maltoporin. Fitting performed well in detergent, lipids, or solution, offering simpler options for fully automated simulation protocols. We were also able to apply fitting to adjust the microscopes pixel size. The approach described here should be applicable to rapid, accurate refinement of a variety of membrane-protein structures.

biophysics↗

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↗

bioRxiv2021

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

biophysics↗