The authors have declared that no competing interests exist.
Conceived and designed the experiments: AB ACK MSPS. Performed the experiments: AB. Analyzed the data: AB ACK. Contributed reagents/materials/analysis tools: ACK. Wrote the paper: AB ACK MSPS.
Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.
Neuromuscular junction formation and maintenance is an essential biological process, the disruption of which leads to congenital myasthenic syndromes and premature death. Dok7 is a key member in formation, maintenance and signaling in neuromuscular junctions. Dok7 is a peripheral membrane protein that is necessary for full activation of the receptor tyrosine kinase MuSK, a receptor tyrosine kinase residing in the postsynaptic membrane. The structure of Dok7 consists of both a PH domain and a PTB domain. The interaction of Dok7 with cell membranes is not well understood. Here, we use molecular simulations to show that the Dok7 PH domain preferentially binds to PIP lipid molecules when associating with a membrane. Dok7 interacts with the bilayer in both a
Dok7 (Downstream-of-Kinase 7) is a peripheral membrane protein and a member of the Dok family of proteins [
PH domains are a structurally conserved family which have been shown to associate with phosphatidylinositol phosphates (PIPs; [
Understanding the mechanism of association of the Dok7 PH domain with a model cell membrane will provide insights into the interactions stabilizing the Dok7/MuSK/membrane complex, since the PH orientation relative to the membrane is likely to play a key role in the Dok7/MuSK interaction. It has been shown that molecular dynamics (MD) simulations provide a tool for understanding the mechanism of association of peripheral proteins with models of cell membranes [
In the current study, we employ a multi-scale MD simulation approach [
We first explored the association of the PH domain of Dok7 with a PIP-containing bilayer. In these simulations, the Dok7 PH domain was initially positioned ~7 nm away from a preformed lipid bilayer composed of 75% PC + 20% PS + 5% PIP2. An ensemble of 20 simulations each of duration 1 μs (see
Simulation | Lipid Bilayer | Duration |
---|---|---|
CG-PH-pip2 | PC/PS/PIP2 (75%/20%/5%) | 20 x 1 μs |
CG-PH-pip3 | PC/PS/PIP3 (75%/20%/5%) | 20 x 1 μs |
CG-PH-pc | PC (100%) | 20 x 1 μs |
CG-PH-pcps | PC/PS (80%/20%) | 20 x 1 μs |
AT-PH-pip2 | PC/PS/PIP2 (75%/20%/5%) | 3 x 0.3 μs |
AT-PH-pip3 | PC/PS/PIP3 (75%/20%/5%) | 3 x 0.3 μs |
Summary of the simulations used in this study. The PIP2 simulations used PI(4,5)P2 (
During each simulation, the protein diffused in the aqueous solution before interacting with the bilayer (
To further analyse the orientation and interactions of the Dok7 PH domain relative to different lipid bilayers, we have constructed two-dimensional density landscapes of the
Analysis of the orientation of the Dok7 PH domain relative to a PIP2-containing membrane reveals that there is a clearly preferred binding mode of the PH domain to the bilayer (M1 in
Densities of the PH domain binding to PIP2
The presence of two PIP-binding sites in the ASAP1 crystal structure is very similar to the arrangement of bound PIP2 molecules we see in the Dok7 simulations (
Alignment of the Arf GAP ASAP1 (pdb id 5C79) with a representative structure from the simulation of the Dok7 PH domain in the presence of PIP2. The Dok7 PH domain is in yellow cartoon, and the PIP2 lipids from the simulation are in purple licorice and CPK. The Arf GAP ASAP1 backbone is in ochre cartoon, and the dibutyryl PIP2 molecules in the crystal structure are in CPK magenta.
To further quantify the similarities between the interactions of the Dok7 PH and ASAP1 PH domain with PIPs, 0.3 μs atomistic simulations of the Dok7 PH domain (see below) were compared to 0.5 μs simulations of the ASAP1 PH domain (the latter are described in detail in [
Evaluation of radial distribution functions (RDFs) of the different lipid species around the bound Dok7 PH domain (
The average, over the 20 repeat simulations, radial distribution function of the coarse-grained simulations with PIP lipids. Averages for the PC/PS lipids were taken from the PC/PS/PIP2 simulations, whilst the PIP3 line is taken from the PC/PS/PIP3 simulations. The PC line is red, PS in green, PIP2 in purple, and PIP3 in orange.
The preferred (i.e. most frequently observed) orientations of the PH domain when bound to either PIP2 or PIP3 as identified from the CG simulations were converted to atomistic representations and multiple (3 x 0.3 μs) atomistic simulations (
We have performed simulations of the Dok7 PH domain with complex lipid bilayers containing PIP2 and PIP3 molecules. Our simulations have shown that PIP molecules were needed for the formation of a stable Dok7 PH/bilayer complex. Furthermore, binding of the PH domain to the bilayer was via interactions of the β1/β2 and β3/β4 loops, i.e. in the region that was previously proposed by Bergamin et al. [
Calculation of density landscapes allowed us to quantify the Dok7 PH domain binding to the bilayer. Our analysis suggests that in the presence of PIP2 molecules in the bilayer, the Dok7 PH domain prefers to bind in the
Recently, a crystal structure of the PH domain of the Arf GAP ASAP1 was solved in complex with dibutyryl-PI(4,5)P2 lipids [
In summary, the PH domain of Dok7 associates with the bilayer via a canonical binding mode observed for other PH domains, as evidenced by the two-dimensional density landscapes we have calculated. By virtue of possessing two binding sites for PIPs, Dok7 causes clustering of PIP lipids around it, potentially facilitating its interaction with MuSK enabling downstream signaling transduction. The simulation methodology employed in this work may be readily applied to other peripheral membrane proteins. Alongside methods such as pMD-Membrane [
The published structure of Dok7 (PDB id: 3ML4) was used as a starting point, using Modeller 9v8 [
CG-MD simulations were performed using the MARTINI2.1 forcefield [
All CG-MD simulations were run with GROMACS 4.5.5 (
To construct these density landscapes, we have merged all 20 simulations for each system. A two-dimensional histogram of
Representative CG snapshots were chosen which corresponded to the distance and
Images of the coarse-grained models of the PI(4,5)P2
(TIF)
Density landscapes for the CG-PH-pip2 system when it is separated into 4 x 5 μs, 2 x 10 μs and 1 x 20 μs densities.
(TIF)
PIP binding sites observed in atomistic simulations of
(TIF)
Number of contacts of Dok7’s PH domain with the headgroups
(TIF)
(TIF)
Hydrogen bonds between the Dok7 PH domain with
(TIF)
Hydrogen bonds of selected residues (K18, K20, R22 and R54) on the Dok7 PH domain with PIP2
(TIF)
Atomistic lipid contacts broken down into headgroup
(TIF)
PDB file of the Dok7 PH domain with a PC/PS/PIP2 bilayer that is aligned with the Arf GAP ASAP1 structure.
(PDB)
We thank Dr. Eiji Yamamoto for providing access to his simulations of ASAP1 for comparison to Dok7.