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Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain. Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.

Molecular simulations reveal that multiple lipid interactions are needed for high-affinity binding of protein domains to membranes., Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

Pleckstrin homology (PH) domains mediate protein-membrane interactions by binding to phosphatidylinositol phosphate (PIP) molecules. The structural and energetic basis of selective PHPIP interactions is central to understanding many cellular processes, yet the molecular complexities of the PH-PIP interactions are largely unknown. Molecular dynamics (MD) simulations using a coarse-grained model enables estimation of free energy landscapes for the interactions of 12 different PH domains with membranes containing PIP2 or PIP3, allowing us to obtain a detailed molecular energetic understanding of the complexities of the interactions of the PH domains with PIP molecules in membranes. Distinct binding modes, corresponding to different distributions of cationic residues on the PH domain, were observed, involving PIP interactions at either the ‘canonical’ (C) and/or ‘alternate’ (A) sites. PH domains can be grouped by the relative strength of their C- and A-site interactions, revealing that a higher affinity correlates with increased C-site interactions. These simulations demonstrate that simultaneous binding of multiple PIP molecules by PH domains contributes to high affinity membrane interactions, informing our understanding of membrane recognition by PH domains in vivo.

Understanding the energetics of peripheral protein-membrane interactions is important to many areas of biophysical chemistry and cell biology. Estimating free-energy landscapes by molecular dynamics (MD) simulation is challenging for such systems, especially when membrane recognition involves complex lipids, e.g., phosphatidylinositol phosphates (PIPs). We combined coarse-grained MD simulations with umbrella sampling to quantify the binding of the well-explored GRP1 pleckstrin homology (PH) domain to model membranes containing PIP molecules. The experimentally observed preference of GRP1-PH for PIP3 over PIP2 was reproduced. Mutation of a key residue (K273A) within the canonical PIP-binding site significantly reduced the free energy of PIP binding. The presence of a noncanonical PIP-interaction site, observed experimentally in other PH domains but not previously in GRP1-PH, was also revealed. These studies demonstrate how combining coarse-grained simulations and umbrella sampling can unmask the molecular basis of the energetics of interactions between peripheral membrane proteins and complex cellular membranes.

Many signaling and trafficking events in cells are regulated by the binding of peripheral membrane proteins to specific lipids (e.g. phosphatidylinositol phosphates; PIPs) in cell membranes. This binding frequently occurs via lipid-binding modules such as the Pleckstrin Homology (PH) domain present in many peripheral proteins. It remains challenging to investigate the molecular mechanisms and the energetics by which these lipid-binding modules associate with lipid membranes. Here we will discuss the application of a high-throughput molecular dynamics simulation approach to investigate the association of peripheral membrane proteins with lipid bilayers of differing phospholipid composition. This computational approach was designed to perform large number of coarse-grained molecular dynamics simulation. It therefore enables detailed investigation of the molecular mechanisms by which peripheral membranes proteins associate with model membranes and construction of the energy landscapes for their binding to model membrane surfaces. In particular, we have determined the mechanism of localization of different PH domains to a PIP-containing bilayer and we have examined the extent of conservation/variation of the interactions of different members of the PH domain family with the lipids. The PH/PIP complexes obtained from our simulations are in good agreement with available structural data. By applying a potential of mean force approach we can also calculate the energy of binding of a PH domain to a PIP-containing bilayer. Additionally, we have extended our studies to other peripheral membrane proteins that contain FERM, PH, and C2 lipid-binding domains. Our results suggest novel models for the association and interactions of these lipid-binding domains with membranes.

Interactions of peripheral membrane proteins with membranes, often mediated by lipid-binding modules such as Pleckstrin Homology (PH) domains, are vital for many signal transduction and related processes in cells. PH domains have been shown to associate with phosphatidylinositol phosphate (PIP) lipids, binding to different PIP species with differing affinities and selectivity. However, the exact molecular and energetic details of the PH-membrane interactions remain in many cases elusive. Potential of mean force calculations, using an umbrella sampling approach, allow quantification of binding affinities. The umbrella sampling methodology has previously been largely limited to smaller systems, e.g. the binding of short peptides to proteins. In this study we have used coarse-grained molecular dynamics simulations and an umbrella sampling approach to quantify the binding of PH domains to model membranes containing PIP lipids, considering particularly the PH domain of GRP1. Out results show that the binding affinity of the GRP1-PH domain for PIP3 molecules in a lipid bilayer is greater than for PIP2, in line with previous studies, while other phospholipids did not exhibit favourable binding. Mutation of four key basic residues in the binding site reduced the affinity of GRP1-PH for PIP3. Analysis of the simulations shows that in addition to the canonical orientation, GRP1-PH can bind to the membrane in a secondary orientation via a separate PIP interaction site. This secondary site coincides with an ‘atypical’ site identified in several other PH domains. These results demonstrate how an umbrella sampling approach, combined with coarse-grained simulations, can be applied to peripheral protein-membrane systems in order to increase understanding of the energetics and molecular details of protein-lipid interactions.