The Role of C2 Domains in Two Different Phosphatases: PTEN and SHIP2.

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.

Specific interactions of peripheral membrane proteins with lipids: what can molecular simulations show us?

Peripheral membrane proteins (PMPs) can reversibly and specifically bind to biological membranes to carry out functions such as cell signalling, enzymatic activity, or membrane remodelling. Structures of these proteins and of their lipid-binding domains are typically solved in a soluble form, sometimes with a lipid or lipid headgroup at the binding site. To provide a detailed molecular view of PMP interactions with the membrane, computational methods such as molecular dynamics (MD) simulations can be applied. Here, we outline recent attempts to characterise these binding interactions, focusing on both intracellular proteins, such as phosphatidylinositol phosphate (PIP)-binding domains, and extracellular proteins such as glycolipid-binding bacterial exotoxins. We compare methods used to identify and analyse lipid-binding sites from simulation data and highlight recent work characterising the energetics of these interactions using free energy calculations. We describe how improvements in methodologies and computing power will help MD simulations to continue to contribute to this field in the future.

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations.

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 µm2 in the search for a second endosome with which to interact.

Large scale model lipid membrane movement induced by a cation switch.

A biomembrane sample system where millimolar changes of cations induce reversible large scale (≥ 200 Å) changes in the membrane-to-surface distance is described. The system composes of a free-floating bilayer, formed adjacent to a self-assembled monolayer (SAM). To examine the membrane movements, differently charged floating bilayers in the presence and absence of Ca2+ and Na+, respectively, were examined using neutron reflectivity and quartz crystal microbalance measurements, alongside molecular dynamics simulations. In neutron reflectivity the variation of Ca2+ and Na+ concentration enabled precision manipulation of the membrane-to-surface distance. Simulations suggest that Ca2+ ions bridge between SAM and bilayer whereas the more diffuse binding of Na+, especially to bilayers, is unable to fully overcome the repulsion between anionic floating bilayer and anionic SAM. Reproduced neutron reflectivity results with quartz crystal microbalance demonstrate the potential of this easily producible sample system to become a standard analysis tool for e.g. investigating membrane binding effects, endocytosis and cell signaling.