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

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

Continuous distribution model for the investigation of complex molecular architectures near interfaces with scattering techniques

Biological membranes are composed of a thermally disordered lipid matrix and therefore require non-crystallographic scattering approaches for structural characterization with x-rays or neutrons. Here we develop a continuous distribution (CD) model to refine neutron or x-ray reflectivity data from complex architectures of organic molecules. The new model is a flexible implementation of the composition-space refinement of interfacial structures to constrain the resulting scattering length density profiles. We show this model increases the precision with which molecular components may be localized within a sample, with a minimal use of free model parameters. We validate the new model by parameterizing all-atom molecular dynamics (MD) simulations of bilayers and by evaluating the neutron reflectivity of a phospholipid bilayer physisorbed to a solid support. The determination of the structural arrangement of a sparsely-tethered bilayer lipid membrane (stBLM) comprised of a multi-component phospholipid bilayer anchored to a gold substrate by a thiolated oligo(ethylene oxide) linker is also demonstrated. From the model we extract the bilayer composition and density of tether points, information which was previously inaccessible for stBLM systems. The new modeling strategy has been implemented into the ga_refl reflectivity data evaluation suite, available through the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR).</p

Parameters of the best-fit model for the neutron reflection from stBLMs with associated PTEN proteins.

<p>Error limits indicate 68% confidence intervals derived from Monte-Carlo data resampling of the data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591-Heinrich1" target="_blank">[39]</a>.</p

Quantification of PTEN Binding to stBLMs by Neutron Reflection.

<p>Exemplary NR data set showing the changes of the neutron reflection upon <i>wt</i> PTEN association with a preformed stBLM composed of DOPC∶DOPS 70∶30, the residuals which emphasize these changes (bottom), and the corresponding nSLD profiles (inset). The NR spectra (main panel) for the stBLM before (black) and after incubation with 20 µM <i>wt</i> PTEN (red) are normalized to the Fresnel reflectivity—<i>i.e.</i>, the reflectivity of a neat Si/buffer interface without interfacial roughness—in order to emphasize the interference patterns due to the interfacial structures. Changes of the spectra upon PTEN association with the membrane are shown as residuals, normalized to the magnitude of the experimental errors, at the bottom. Lines in the main panel show the computed NR of the nSLD profiles shown in the inset. Note that these profiles are derived by fitting multiple data sets simultaneously (neat stBLM and stBLM with PTEN, each measured at different isotopic buffer contrasts) by sharing model parameters as appropriate. A dashed box in the inset indicates the region of the distal lipid headgroups and associated PTEN protein, shown in close-up view in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone-0032591-g004" target="_blank">Fig. 4</a>. The signal-to-noise in these measurements is comparable to that in similar studies on the incorporation of α-hemolysin into stBLMs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591-McGillivray2" target="_blank">[33]</a> and the binding of the HIV-1 matrix protein to bilayer surfaces <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591-Nanda1" target="_blank">[34]</a>.</p

Quantification of mutant PTEN Binding to stBLMs by SPR.

<p>Dissociation constants, <i>K_d</i>, in µM for the binding, at room temperature, of <i>wt</i> PTEN to stBLMs prepared by RSE on SAMs composed of HC18∶βME 70∶30 from lipid solutions of the compositions shown. The lipid chain compositions are dioleoyl (DOPC, DOPS) and that of the natural brain extract for PI(4,5)P₂ (mostly stearoyl-arachidonoyl). All experiments were evaluated as single component fits. The parameter ranges given for adsorption of the truncated PTEN PC∶PS stBLMs were estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591.s001" target="_blank">Information S1</a>.</p

Schematic Depiction of the PTEN Phosphatase on the Surface of a Thermally Disordered stBLM.

<p>Peptide backbone representation of (A) <i>wt</i> PTEN and (B) H93R PTEN positioned at a DOPC/DOPS (7∶3) membrane surface as deducted from the NR results. The membrane-associated protein penetrates the lipid headgroups (PC: violet, PS: orange) only barely. The PTEN core domains (PD: magenta, C2: grey) are shown in a conformation and membrane orientation deduced from the crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591-Lee1" target="_blank">[24]</a>. The close correspondence, observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone-0032591-g004" target="_blank">Fig. 4</a>, between the nSLD distribution across the interface determined in this work and the nSLD distribution of the truncated PTEN computed from the crystal structure suggests that this is a good approximation. Moreover, about 20% of the protein mass have been deleted in the truncated protein, and ∼20% of the nSLD remains unaccounted for in the overall nSLD distribution for <i>wt</i> PTEN in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone-0032591-g004" target="_blank">Fig. 4A</a>, if we position the PTEN core domains at the membrane as shown here. The C-terminal tail, which forms the bulk of the deleted peptide, is apparently quite different in its organization in <i>wt</i> and H93R PTEN at the membrane. Shown here in red, yellow and green are three distinct conformations, obtained from Monte-Carlo simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone.0032591-Curtis1" target="_blank">[57]</a>, on each PTEN protein core that are consistent with the observed nSLD distributions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032591#pone-0032591-g004" target="_blank">Figs. 4A and C</a>.</p