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KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, K

KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, NMR, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.

Interfaces between molecular organic architectures and oxidic substrates are a central feature of biosensors and applications of biomimetics in science and technology. For phospholipid bilayers, the large range of pH- and ionic strength-dependent surface charge densities adopted by titanium dioxide and other oxidic surfaces leads to a rich landscape of phenomena that provides exquisite control of membrane interactions with such substrates. Using neutron reflectometry measurements, we report sharp, reversible transitions that occur between closely surface-associated and weakly coupled states. We show that these states arise from a complex interplay of the tunable length scale of electrostatic interactions with the length scale arising from other forces that are independent of solution conditions. A generalized free energy potential, with its inputs only derived from established measurements of surface and bilayer properties, quantitatively describes these and previously reported observations concerning the unbinding of bilayers from supporting substrates.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

A framework is applied to quantify information gain from neutron or X-ray reflectometry experiments [Treece, Kienzle, Hoogerheide, Majkrzak, Lösche & Heinrich (2019). J. Appl. Cryst. 52, 47–59], in an in-depth investigation into the design of scattering contrast in biological and soft-matter surface architectures. To focus the experimental design on regions of interest, the marginalization of the information gain with respect to a subset of model parameters describing the structure is implemented. Surface architectures of increasing complexity from a simple model system to a protein–lipid membrane complex are simulated. The information gain from virtual surface scattering experiments is quantified as a function of the scattering length density of molecular components of the architecture and the surrounding aqueous bulk solvent. It is concluded that the information gain is mostly determined by the local scattering contrast of a feature of interest with its immediate molecular environment, and experimental design should primarily focus on this region. The overall signal-to-noise ratio of the measured reflectivity modulates the information gain globally and is a second factor to be taken into consideration.

Src family kinases (SFKs) are a group of non-receptor tyrosine kinases that are characterized by their involvement in critical signal transduction pathways. SFKs are often found attached to membranes but little is known about the conformation of the protein in this environment. Here, solution nuclear magnetic resonance (NMR), neutron reflectometry (NR), and molecular dynamics (MD) simulations were employed to study the membrane interactions of the intrinsically disordered SH4 and Unique domains of the Src family kinase Hck. Through development of a procedure to combine the information from the different techniques, we were able produce a first-of-its-kind atomically detailed structural ensemble of a membrane-bound intrinsically disordered protein. Evaluation of the model demonstrated its consistency with previous work and provided insight into how SFK Unique domains act to differentiate the family members from one another. Fortuitously, the position of the ensemble on the membrane allowed the model to be combined with configurations of the multi-domain Hck kinase previously determined from small-angle solution X-ray scattering to produce full-length models of membrane-anchored Hck. The resulting models allowed us to estimate that the kinase active site is positioned about 65 ± 35 Å away from the membrane surface, offering the first estimations of the lengthscale associated with the concept of SFK subcellular localization.

A framework for the optimization of neutron reflectometry experiments based on Bayesian statistics and information theory is presented., A framework based on Bayesian statistics and information theory is developed to optimize the design of surface-sensitive reflectometry experiments. The method applies to model-based reflectivity data analysis, uses simulated reflectivity data and is capable of optimizing experiments that probe a sample under more than one condition. After presentation of the underlying theory and its implementation, the framework is applied to exemplary test problems for which the information gain ΔH is determined. Reflectivity data are simulated for the current generation of neutron reflectometers at the NIST Center for Neutron Research. However, the simulation can be easily modified for X-ray or neutron instruments at any source. With application to structural biology in mind, this work explores the dependence of ΔH on the scattering length density of aqueous solutions in which the sample structure is bathed, on the counting time and on the maximum momentum transfer of the measurement. Finally, the impact of a buried magnetic reference layer on ΔH is investigated.

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζcyt) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζcyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζcyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζcyt to the bilayers in dynamic equilibrium: one in which ζcyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζcyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Efficient assembly of HIV-1 at the plasma membrane (PM) of the T-cell specifically requires PI(4,5)P2. It was previously shown that a highly basic region (HBR) of the matrix protein (MA) on the Gag precursor polyprotein Pr55Gag is required for membrane association. MA is N-terminally myristoylated, which enhances its affinity to membranes. In this work we used X-ray scattering and neutron reflectivity to determine how the physical properties and structure of lipid bilayers respond to the addition of binding domain peptides, either in the myristoylated form (MA31myr) or without the myristoyl group (MA31). Neutron reflectivity measurements showed the peptides predominantly located in the hydrocarbon interior. Diffuse X-ray scattering showed differences in membrane properties upon addition of peptides and the direction of the changes depended on lipid composition. The PI(4,5)P2-containing bilayers softened, thinned and became less ordered as peptide concentration increased. In contrast, POPS-containing bilayers with equivalent net charge first stiffened, thickened and became more ordered with increasing peptide concentration. As softening the host cell’s PM upon contact with the protein lowers the free energy for membrane restructuring, thereby potentially facilitating budding of viral particles, our results suggest that the role of PI(4,5)P2 in viral assembly goes beyond specific stereochemical membrane binding. These studies reinforce the importance of lipids in virology.

We present a novel method to incorporate structural results from surface-sensitive scattering, such as X-ray or neutron reflectometry, into molecular dynamics simulations. While reflectometry techniques generally provide a means to determine the molecular-scale structures of organized interfacial films, they were recently shown to offer the capability to characterize the structures of protein-membrane complexes supported by a solid substrate. One-dimensional information inherent in the experimental results is used in the form of component volume occupancy (CVO) profiles, which describe the distribution of molecular components within an interfacial architecture, to construct real-space constraints in the form of a biasing potential for the simulation that vanishes when the simulated and experimental profiles agree. This approach improves the correspondence between simulation and experiment, as shown in the re-evaluation of an neutron-reflection-derived structure which was approximated by an independent molecular dynamics simulation in earlier work, and it also leads to faster equilibration of ensemble structures. We further show that time averaging the CVO profile that develops in the simulation while biasing with this approach permits fluctuations about the average that are necessary for conformational exploration of the system. This method is particularly valuable for studies of proteins at interfaces that contain disordered regions since the conformation of such regions is difficult to judge from the analysis of one-dimensional experimental profiles and may take prohibitively long to equilibrate in simulations.

By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, Δ G , of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P 2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P 2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P 2 , the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P 2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.

International audience; The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. Published version

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions (Shenoy et al., 2012, PLoS ONE 7, e32591) and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN's C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN's C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN's unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN's membrane binding and activity.

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001, eLife digest Streptococcus pyogenes is the bacterium that causes throat infections and other serious infections in humans. Antibiotics such as penicillin are used to treat active infections, but so-called “strep throat infections” often return after treatment. This is because S. pyogenes can enter the cells that line the throat and hide from the antibiotics, which cannot enter the throat cells. Endolysins are enzymes produced by viruses that attack bacteria, and these enzymes target and destroy the bacterial cell wall. A previous study revealed that an endolysin known as PlyC could destroy S. pyogenes bacteria on contact. PlyC and other endolysins have the potential to act as alternatives to common antibiotics, but before these enzymes can be developed as therapeutics, it is important to understand how they interact with human host cells. Like antibiotics, the PlyC endolysin was not expected to enter throat cells. However, Shen, Barros et al. have now discovered that not only can PlyC enter throat cells, it can essentially chase down and kill S. pyogenes that are hiding inside. Other similar enzymes could not act in this way, and further studies confirmed that PlyC could move around inside a throat cell without causing it damage. Shen, Barros et al. also determined that PlyC has a pocket on its surface that binds with a specific component of the throat cell membrane, a molecule called phosphatidylserine. This interaction – which is a bit like a lock and key – grants PlyC access into the cell. While it is clear that PlyC eventually kills S. pyogenes hiding inside throat cells, future experiments will aim to determine how PlyC moves around once inside an infected throat cell. Together, an understanding of how an endolysin enters cells and destroys hiding S. pyogenes will contribute to the development of endolysins with broader activity, which can be used as alternatives to common antibiotics. DOI: http://dx.doi.org/10.7554/eLife.13152.002

The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo , plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.

The Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases known for their involvement in critical signal transduction pathways. Structurally, SFKs share a common multi-domain architecture, consisting of SH3, SH2, and catalytic (SH1) domains attached to a membrane-anchoring SH4 domain through an intrinsically disordered ∼80 residue region of low sequence conservation called the Unique (U) domain. Despite the wealth of information on SFK structure and function, little is known about the nature of these enzymes in the membrane-associated form. Interestingly, SH4-U domains in different species share common sequence features, despite differences between SFK members, arguing for their importance in differentiating the functions of SFKs and raising questions about their ability to participate in SFK signaling.Hematopoetic cell kinase (Hck), a phagocyte specific proto-oncogene member of the SFKs, is a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. To achieve structural characterization of the membrane-associated Hck via its SH4-U domains, data from solution and solid-state NMR, neutron reflection, and fluorescence resonance energy transfer were computationally integrated using a novel maximum-entropy multi-resolution “restrained-ensemble molecular dynamics” (reMD) simulation scheme. This framework has allowed us to detail features of the membrane-protein complex at atomic resolution and identify possible new constraints on Hck's ability to phosphorylate downstream targets. In vivo assays are being developed to test the relevance of these findings.

We demonstrate a method for simultaneous structure and function determination of integral membrane proteins. Electrical impedance spectroscopy shows that Staphylococcus aureus α-hemolysin channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry provides high-resolution structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channel's x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 Å. The channel's extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate molecular details of the association of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.

Tethered bilayer lipid membranes have been shown to be an excellent model system for biological membranes. Coupling of a membrane to a solid supports creates a stable system that is accessible for various surface analytical tools. Good electrical sealing properties also enable the use of the membranes in practical sensing applications. The authors have shown that tethered membranes have extended lifetimes up to several months. Air-stability of the bilayer can be achieved by coating the membrane with a hydrogel. The structure of a monolayer and its stability under applied dc potentials have been investigated by neutron scattering. © 2008 American Vacuum Society.

Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochemical impedance spectroscopy, neutron reflection (NR), and Fourier-transform infrared reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that electrically insulating membranes (conductance and capacitance as low as 1 microS cm(-2) and 0.6 microF cm(-2), respectively) with high surface coverage (95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by "rapid solvent exchange." NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain "backfiller," beta-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by volume, as judged from NR and contrast variation of the solvent. Complete and stable "sparsely tethered" BLMs (stBLMs) can be readily prepared from SAMs chemisorbed from solutions with low WC14 proportions. Phospholipids with unsaturated or saturated, straight or branched chains all formed qualitatively similar stBLMs.

SummaryAs the phosphoinositol-3-kinase antagonist in the PI3K pathway, the PTEN tumor suppressor exerts phosphatase activity on diacylphosphatidylinositol triphosphate in the plasma membrane. Even partial loss of this activity enhances tumorigenesis, but a mechanistic basis for this aspect of PTEN physiology has not yet been established. It was recently proposed that PTEN mutations have dominant-negative effects in cancer via PTEN dimers. We show that PTEN forms homodimers in vitro, and determine a structural model of the complex from SAXS and Rosetta docking studies. Our findings shed new light on the cellular control mechanism of PTEN activity. Phosphorylation of the unstructured C-terminal tail of PTEN reduces PTEN activity, and this result was interpreted as a blockage of the PTEN membrane binding interface through this tail. The results presented here instead suggest that the C-terminal tail functions in stabilizing the homodimer, and that tail phosphorylation interferes with this stabilization.

The PTEN tumor suppressor is a dual-specificity phosphatase whose main target is the phosphatidylinositoltriphosphate (PI(3,4,5)P3) pool in the inner plasma membrane. It acts as the PI3K antagonist in the PI3K/Akt signaling pathway that controls cell growth and apoptosis and is the second most frequently mutated protein in human cancers. Because the propensity for tumor formation depends on PTEN dose in a way that is inconsistent with the “two-hit hypothesis”, it was postulated that PTEN multimers may form the active species of the phosphatase, and recently strong evidence has been presented for the formation of functional PTEN dimers in the cell (Papa et al., Cell 157, 2014, 595). Here, we use SAXS to investigate the multimerization of PTEN in buffer and show that it indeed forms dimers following elution from a size-exclusion column as a monomer. Electron density envelopes for the PTEN monomer and dimer obtained from SAXS could clearly be distinguished and were assigned by placing the PTEN crystal structure which was earlier determined for a truncated protein. In addition, the monomer envelope was validated by μs-long all-atom MD simulations of full-length PTEN. In these simulations, the auto-inhibitory, flexible C-terminal tail associates closely with the PTEN core domains while hopping between different bound conformations. The equilibrium ensemble of the resulting structures is in excellent agreement with the SAXS data. A structure prediction using the Rosetta docking protocol revealed a putative dimer arrangement that fits the dimer envelope derived from SAXS very well and is consistent with neutron reflectometry results for membrane-bound PTEN.

The rate of Ca2+ -triggered phospholipase A2 (PLA2) degradation of tethered bilayer membranes (tBLMs), composed of a synthetic lipid, beta-mercaptoethanol, and palmitoyloleoylphosphatidylcholine (POPC), is approximately 80 times greater than for those prepared with diphytanoylphosphatidylcholine (DPhyPC). Electrochemical impedance spectroscopy (EIS) and neutron reflectivity (NR) data indicate complete, water-free tBLMs that exhibit near ideal capacitive behavior and the presence of a water reservoir in the bilayer subspace proximal to the substrate (Au) surface for both tBLMs. Together these data indicate that the POPC and the DPhyPC tBLMs are structurally similar along the surface normal but markedly different at the outer leaflet/solution interface and that PLA2 is a sensitive probe of short length scale structural differences not revealed by EIS and NR.

Subtle differences in the molecular conformation of fully hydrated phospholipids, and in their interaction with the water reservoir, were assessed as functions of headgroup methylation with surface-sensitive X-ray scattering. To achieve such a structural and functional comparison, diacylphosphatidylethanolamines (PEs) and their mono-, di- and trimethylated (diacylphosphatidylcholine, PC) derivatives in surface monolayers on water have been studied. While the molecular structures of these lipids are quite similar, their subtle distinctions lead to surprisingly large differences in their overall organization. Independent of the surface pressure, pi, the amine function in PE extends 1-2 A further into the subphase than those of the methylated headgroups. Not only is the exposure of the amine moiety to water in PE thus larger than that of the other lipids, but also the phosphate and lipid backbone of PE are more hydrated than that of PC. Overall, the PE headgroup hydration is approximately 25% larger than that of PE-N-Me, PE-N-Me2 or PC. The main reason for these differences resides in their distinct capabilities to donate hydrogen bonds, but differences in the hydrophobicities of the amine functions on the lipid headgroups may also play a role. While the impact of amine methylation on the headgroup interaction with the water subphase appears rather straightforward, there are also differences in lipid backbone organization and acyl chain packing. The results presented here provide a deeper understanding of lipid conformation as the hydrophobicity of the terminal headgroup fragment is systematically altered and may also impact on our understanding of the molecular details of membrane fusion.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases expressed during the late stages of a phage replication cycle. that function to lyse the bacterial cell wall, thus enabling progeny phage release. When exogenously added, these enzymes lyse the peptidoglycan of Gram-positive pathogens, resulting in osmotic lysis and cell death. One particular endolysin, PlyC, gains cellular entry and clears intracellular streptococci without compromising cell viability, implying that an unknown mechanism provides entry to the streptococci-specific endolysin into mammalian cells. Here, we investigate the molecular basis of PlyC binding domain, PlyCB, ability to bind and cross the mammalian plasma membrane using synthetic model membranes.Complementary surface-sensitive techniques showed membrane integrity during PlyCB exposure and quantified membrane-binding affinity. Surface plasmon resonance data reveals that while the interaction of PlyCB with purely zwitterionic membranes is negligible, the protein strongly interacts with anionic membranes that contain phosphatidylserine (PS) above a well-defined concentration threshold. In contrast, PlyCB affinity for other anionic lipids tested is low, suggesting specificity for PS. Neutron reflection data showed that PlyCB binding to the membrane surface is followed by penetration into the hydrophobic membrane core, while impedance spectroscopy confirms that the membrane integrity is not affected. Those findings provide clues to a mechanistic understanding of how PlyCB binds and initiates membrane translocation. After the initial membrane interaction, PlyCB is known to subsequently internalize and be trafficked inside the cell where it eventually kills the streptococci. PlyC's entry point into the cell, PS is primarily located in the inner leaflet of the plasma membrane where it would not be available for binding external endolysin. However, in cells stressed by bacteria assault and inflammation, lipid asymmetry is compromised: PS may be exposed on the outer plasma membrane.

Langmuir monolayers of 4'-(4-methylphenyl)-5'-phenyl-1,1':3',1"-terphenyl-4-carboxylic acid (p-tolyl-PTCA) show a well-defined surface isotherm indicative of a fluid phase followed by a pronounced and broad plateau in which the molecular area decreases by about a factor of 2. The origin of this plateau has been investigated using fluorescence and Brewster angle microscopy (BAM) as well as Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). Whereas the surface structure is monomolecular at pressures below the onset of the plateau, the experimental results point to the formation of structures that are no longer monomolecular in the plateau region: As the plateau is reached, changes in the film structure are observed in fluorescence micrographs whereas FT-IRRAS indicates a change in the hydration of the carboxyl groups. A 4-fold increase in reflectivity observed in BAM across the plateau corresponds to an increase in film thickness by a factor of 2. This may correspond to the formation of a laterally homogeneous double layer or to a surface structure that is inhomogeneous on a submicrometer length scale, in which monolayer regions coexist with areas covered with more than two monolayers of material. Subsidiary AFM results indicate the presence of triple-layer patches scattered randomly over a smooth monolayer along the plateau. This is in contradiction to the earlier hypothesis of molecular tilting, which was thought to be more probable than a multilayer formation (Dynarowicz-Łątka, P.; Dhanabalan, A.; Cavalli, A.; Oliveira, O. N., Jr. J. Phys. Chem. B 2000, 104, 1701). It thus appears that although polyphenyl carboxylic acids (PTCA and its derivatives) do not show liquid crystalline properties their surface structure resembles more closely that of molecular mesogen films rather than that of monolayers formed by conventional amphiphilic compounds.

Structural details of the coupling of bacterial surface (S)-layers to the phospholipid, dipalmitoylphosphatidylethanolamine (DPPE), have been characterized using X-ray and neutron reflectometry. We studied the binding andrecrystallization of S-protein isolated from B. sphaericus CCM2177 at DPPE monolayers on aqueous surfaces. Particular emphasis has been put on investigations of the lipid/protein interface in a joint refinement of X-ray and neutron data which reveals alterations of the molecular-level organization of the lipid headgroups upon protein binding and recrystallization: Peptide material interpenetrates the phospholipid headgroups almost in its entire depth but does not affect the hydrophobic lipid acyl chains. Consistent with FTIR results, we find that the headgroup hydration is reduced by ∼40% upon peptide interpenetration. On average, the equivalent of ∼65 electrons associated with the peptide, i.e., less than one peptide side group, interacts directly with one DPPE headgroup within the surface film. This suggests that the protein attaches to specific molecular moieties within the lipid monolayer which may form a lateral pattern within the film area that reflects the properties of the monomolecular protein crystal sheet.

The effect of electrostatic dipole interactions on the wetting behavior of two-dimensional droplets, approximated by circular segment shapes with contact angle α, is investigated. α depends on the geometric structure far away from the three-phase contact point. We theoretically find metastable α values associated with transitions from complete wetting to partial dewetting, triggered by minute changes of the film area, the surface potential of the coexisting phases, or their line tensions. The predictions are confirmed experimentally in a Langmuir monolayer model system. The wetting instability may influence intramembrane biochemical reactions between protein species dissolved in coexisting phases via the length of the contact line between the phases, the only region where chemical reactions between the species are possible.

The structural characterization of peripheral membrane proteins represents a tremendous challenge in structural biology due to their transient interaction with the membrane and the potential multitude of protein conformations during this interaction. Neutron reflectometry is uniquely suited to address this problem because of its ability to structurally characterize biological model systems nondestructively and under biomimetic conditions that retain full protein functionality. Being sensitive to only the membrane-bound fraction of a water-soluble peripheral protein, neutron reflectometry obtains a low-resolution average structure of the protein-membrane complex that is further refined using integrative modeling strategies. Here, the authors review the current technological state of biological neutron reflectometry exemplified by a detailed report on the structure determination of the myristoylated human immunodeficiency virus-1 (HIV-1) Gag matrix associated with phosphoserine-containing model membranes. The authors found that the HIV-1 Gag matrix is able to adopt different configurations at the membrane in a pH-dependent manner and that the myristate group orients the protein in a way that is conducive to PIP2-binding.

Human immunodeficiency virus-1 (HIV-1) Nef is a membrane-associated accessory protein essential for viral replication, immune evasion, and AIDs progression. Nef functions via interactions with numerous host cell proteins involved in signal transduction and trafficking. Nef interaction with Src family kinases in HIV-1 target cells leads to constitutive kinase activity that enhances infectivity and viral replication while down-regulating immune receptors. Recently, Nef was also shown to interact selectively with Tec kinases in HIV-1 target cells.

Neutron reflection (NR) from planar interfaces is an emerging technology that provides unique and otherwise inaccessible structural information on disordered molecular systems such as membrane proteins associated with fluid bilayers, thus addressing one of the remaining challenges of structural biology. Although intrinsically a low-resolution technique, using structural information from crystallography or NMR allows the construction of NR models that describe the architecture of protein-membrane complexes at high resolution. In addition, a combination of these methods with molecular dynamics (MD) simulations has the potential to reveal the dynamics of protein interactions with the bilayer in atomistic detail. We review recent advances in this area by discussing the application of these techniques to the complex formed by the PTEN phosphatase with the plasma membrane. These studies provide insights in the cellular regulation of PTEN, its interaction with PI(4,5)P2 in the inner plasma membrane and the pathway by which its substrate, PI(3,4,5)P3, accesses the PTEN catalytic site.

Hutchinson–Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein–membrane association mediated by the retained farnesyl group. To better characterize the protein–membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca[superscript 2 +] or Mg[superscript 2 +] at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association., National Institute of General Medical Sciences (U.S.) (1R01-GM101647), United States. Office of Naval Research. Presidential Early Career Award for Scientists and Engineers (N000141010562), National Institutes of Health (U.S.) (U01 EB014976)