Membrane Association of the PTEN Tumor Suppressor: Molecular Details of the Protein-Membrane Complex from SPR Binding Studies and Neutron Reflection

Abstract

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 µM and 0.4 µM, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

“…The refractive index increment dn/dc Ϸ 0.185 ml/g (35,36). The sensitivity of the instrument is ⌬n ϭ (6.4 Ϯ 0.3) ϫ 10 -5 /RU, where the response unit (RU) is the shift in the SPR reflection minimum on the detector in pixels (37). The SPR responses at high c p were accordingly corrected.…”

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

“…The substrates, No. 1.5 glass coverslips or microscope glass slides for SPR and microscopy, respectively, and 3-inch (75-mm) silicon wafers (El-Cat Inc., Ridgefield Park, NJ) for NR, were thoroughly cleaned as described (21) and metal-coated by magnetron sputtering (ATC Orion, AJA International, Inc., North Scituate, MA). For fluorescence microscopy, ϳ6 nm of gold was sputtered on top of an ϳ2-nm thick chromium bonding layer, which leaves the coverslips optically transparent.…”

“…Neutron Reflection Measurements and Data Modeling-NR measurements were performed on the NG7 and MAGIK reflectometers at the NIST Center for Neutron Research and evaluated with garefl (23) using the GRASP55 crystal structure (Protein Data Bank (PDB): 3RLE), similarly to studies of other membrane-associated proteins (21,24). To determine the protein orientation on the membrane, the origins of the two relevant Euler angles (see Fig.…”

“…The following observations suggest that this is not the case. 1) Structural investigations with NR of other proteins at membrane surfaces, conducted at similar protein surface concentrations (21,34,38), showed clearly that protein organization depended on other factors (electrostatic interaction; ligand binding) than surface crowding. 2) A model that places proteins randomly on the surface (39) predicts mean nearest neighbor distances of 1.6 or 2.25 protein diameters, respectively, for area coverages of 20% (myrG55) or 10% (G55).…”

“…In both methods, the lipid membrane was described as continuous distributions of submolecular fragments (36). The contribution of the membrane-bound protein to the neutron scattering length density at the interface was modeled either as a sequence of spline functions ("model-free description") (21,37) or by using the crystal structure of the GRASP domain (11) by rotating the structural model about two Euler angles and varying its distance from the membrane surface while optimizing the fit to the experimental data (24). Both models allowed for protein penetration into the bilayer, which was found to be small (Ͻ5 Å) in all cases.…”