Search

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

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 cryoelectron 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.

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

AbstractRT-LAMP detection of SARS-CoV-2 has been shown as a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validated the optimized RT-LAMP assay for the detection of SARS-CoV-2 in raw nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings with limited economic resources.

Density guided simulations files .mrc files - CryoEM density half-maps for pH 7, pH 5, pH 3 .mdp - Option files for GROMACS simulations. One density guided mdp file and one annealing mdp file per pH data set .gro - Equilibrated starting model of manually built pH 7,the same was used for all three pH data sets .itp - topology files .top - topology master file Example command for density guided simulations gmx grompp --f mdrun_ip_ph3_tc40_sp02_tau0_3.mdp --p topology.top --o mdrun_ip_ph3_tc40_sp02_tau0_3.tpr --c minimized.gro gmx mdrun --s mdrun_ip_ph3_tc40_sp02_tau0_3_test.tpr --v --x mdrun.xtc --g md.log --e ener.edr --c final_ip_ph3_tc40_sp02_tau0_3_test.gro Example command for custom GROMACS FSC tool, available at (https://github.com/blauc/gromacs/tree/fscavg) gromacs-fsc/bin/gmx fsc -f whole.xtc -s mdrun_ip_ph3_tc40_sp02_tau0_3_test.tpr -refmap pH3_half1_class001_unfil.mrc-dt 100 -xvg none -shells 186 -o fsc_ip_ph3_tc40_sp02_tau0_3.xvg -similarity similarity_ip_ph3_tc40_sp02_tau0_3.xvg -avg fscavg_ip_ph3_tc40_sp02_tau0_3.xvg -fscmove fscmove_ip_ph3_tc40_sp02_tau0_3.xvg -ordinate-axis fscaxis_ip_ph3_tc40_sp02_tau0_3.xvg -fscmoveavg fscmoveavg_ip_ph3_tc40_sp02_tau0_3.xvg Molecular dynamics simulations .prep_file -topology files and forcefield files for preparing the simulations. .run_tpr -tpr files for production runs .prodution_traj -concatenated trajectories and starting snapshots (after equilibration). analysis_results - csv: analaysis data - ipynb: jupyter notebook for generating the figures in the paper.