Dimerization of the EphA1 Receptor Tyrosine Kinase Transmembrane Domain: Insights into the Mechanism of Receptor Activation

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape

Simulation-Based Prediction of Phosphatidylinositol 4,5-Bisphosphate Binding to an Ion Channel

Protein–lipid interactions regulate many membrane protein functions. Using a multiscale approach that combines coarse-grained and atomistic molecular dynamics simulations, we have predicted the binding site for the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) on the Kir2.2 inwardly rectifying (Kir) potassium channel. Comparison of the predicted binding site to that observed in the recent PIP₂-bound crystal structure of Kir2.2 reveals good agreement between simulation and experiment. In addition to providing insight into the mechanism by which PIP₂ binds to Kir2.2, these results help to establish the validity of this multiscale simulation approach and its future application in the examination of novel membrane protein–lipid interactions in the increasing number of high-resolution membrane protein structures that are now available

PIP₂-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP₂) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP₂ molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1−KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP₂-containing lipid bilayers identified the PIP₂-binding site on each channel. These models of the PIP₂−channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP₂ in the atomistic simulations, enabling identification of key side chains

PIP₂-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP₂) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP₂ molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1−KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP₂-containing lipid bilayers identified the PIP₂-binding site on each channel. These models of the PIP₂−channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP₂ in the atomistic simulations, enabling identification of key side chains

Representative data for rich media, 37C

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. This is a raw video file representative of data collected in rich media at 37C (no perturbations). There are two channels: a 'green' channel (488 nm excitation) showing GFP-FtsZ, and a 'red' channel (561 nm excitation) showing single-molecules of HaloTag-PBP2B.</p

Supplementary Video 16 source data

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. 231117_sh203_phmm_30c_flatpad_slide2_008 is the raw video file used to make Svid 16 (Supplementary_Video_16_SFigure17_FtsZ_G106S).</p

Extended Data Figure 9 source data

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. 220303_slide2_5_MMStack_Default_561_denoise.ome_ring6, 220303_slide2_5_MMStack_Default_561_denoise.ome_ring6_kymo, and 230619_220303_slide2_6_ring4_kymo_intensity correspond to ED Fig. 9a. 220303_slide2_6_MMStack_Default_561_denoise.ome_ring4, 220303_slide2_6_MMStack_Default_561_denoise.ome_ring4_kymo, and 230619_220303_slide2_6_ring4_kymo_intensity correspond to ED Fig. 9b. 220309_slide2_4_MMStack_Default_561_denoise.ome_ring9, 220309_slide2_4_MMStack_Default_561_denoise.ome_ring9_kymo, and 230619_220309_slide2_4_ring9_kymo_intensity correspond to ED Fig. 9c. 220310_5_MMStack_Default_561_denoise.ome_ring23, 220310_5_MMStack_Default_561_denoise.ome_ring23_kymo, and 230619_220310_5_ring23_kymo_intensity correspond to ED Fig. 9d.</p

Supplementary Video 2 source data

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. 220316_5_MMStack_Default_561_denoise.ome_ring14 is the raw video file used to produce Svid 2 (Supplementary_Video_2_Figure_1c_bottom)</p

Representative raw data for minimal media, 37C

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. This is a raw video file representative of data collected in minimal media at 37C (no perturbations). There are two channels: a 'green' channel (488 nm excitation) showing GFP-FtsZ, and a 'red' channel (561 nm excitation) showing single-molecules of HaloTag-PBP2B.</p

Supplementary Video 11 source data

This dataset is part of the 'Source data and representative raw data for "Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis"' collection. For a link to the related paper, study-level information and guide to the data, as well as viewing all other related research data, please visit the collection. 220316_3_MMStack_Default_561_denoise.ome_ring11 is the raw video file used to make Svid 11 (Supplementary_Video_11_Figure_4b).</p