The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

ADP ribosylation factor-1 (Arf1) is a major regulator of cell membrane trafficking and organelle structure, and Arf1 misregulation contributes to invasion and metastasis of cancer cells. The basis for Arf1 biological function is a highly regulated GDP/GTP cycle coupled to a cytosolic/membrane-bound cycle. Cell membranes play a crucial role for all steps of the Arf1 signaling cycle: 1) membranes are mandatory for Arf1 GTPase to acquire the active state; 2) Arf1 interacts with numerous proteins on the membrane surface.

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, Kd, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.

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.

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.

Long-term effects of angiotensin converting enzyme inhibitors on split renal function were studied using a noninvasive radioisotopic method in five patients with renovascular hypertension. In the stenotic side the glomerular filtration rate which acutely decreased following captopril, tended to return toward pretreatment levels on prolonged captopril administration (up to 2 years), while the value remained unchanged in the nonstenotic side of the kidney. Effective renal plasma flow increased in both sides. During long-term captopril treatment, there were no significant changes in serum creatinine, sodium, potassium, and blood pressure control was maintained at the same level in an acute period. These results suggest that the glomerular filtration in the stenotic kidney of renovascular hypertension, which was acutely reduced, will not further deteriorate, but will increase after prolonged captopril treatment.

KRAS is a GTPase that plays an important role in cell growth and signaling pathways. of the different RAS isoforms, KRAS also has the highest prevalence of mutations related to human cancers, making it an attractive therapeutic target in these cases. Once attached to the membrane, KRAS in the active (GTP) form is capable to bind effector proteins, like RAF kinase. However, certain molecular details concerning KRAS conformation and orientational changes when interacting with the membrane and binding partners are not fully understood. To provide new insights, we used a variety of biophysical approaches to characterize KRAS structure and dynamics. Here, we focus on our results utilizing data-driven computational docking to investigate both KRAS and KRAS/RAF1-RBD (RAS Binding Domain) complex at the membrane. with the HADDOCK program, we incorporated experimental restraints derived from our NMR paramagnetic relaxation enhancement (PRE) and neutron reflectivity (NR) measurements to dock these KRAS forms to a 70:30 POPC:POPS lipid membrane surface. Using NMR-PRE restraints alone, we performed one series of docking runs with the KRAS G-domain directly interacting with the membrane to discern membrane-proximal states. Based on our experimental evidence, and particularly from NR, a highly populated membrane-distal state also exists, where the G-domain does not directly contact the membrane but KRAS remains tethered via the C-terminal hypervariable region (HVR). Therefore, we also conducted a second series of docking runs that incorporated both NMR-PRE and NR restraints to better elucidate the conformations in this state. From these results, we were able to generate atomistic models for KRAS and KRAS/RAF1-RBD with averaged 1-D profiles closely matching the respective NR profiles. Overall, the findings should assist in elucidating the role of KRAS structural dynamics in recruiting effectors, like RAF kinase, to the membrane for activation.

The guanine nucleotide exchange factor SmgGDS is implicated in a number of oncogenic pathways driven by the Ras family of small GTPases which is involved in 30% of all human cancers. SmgGDS interacts specifically with small GTPases, such as KRas4B, that contain a polybasic region (PBR) and acts as a chaperone that traffics small GTPases to the plasma membrane. Two SmgGDS isoforms are proposed to have differing roles in regulating these oncogenic pathways. SmgGDS-558 has been shown to associate with prenylated small GTPases whereas SmgGDS-607 binds unprenylated small GTPases. Setting out to identify differences in the roles of the two SmgGDS isoforms in Ras regulation, we studied their binding to prenylated KRas4B in solution and on model membranes. Here, we present SAXS studies of SmgGDS-558 and KRas4B in solution which we complement with Rosetta docking simulations and show that prenylated KRas4B binds SmgGDS-558 in a different complex than RhoA for which a crystal structure was recently reported. SPR studies of SmgGDS association with membrane-bound KRas4B show SmgGDS-558 efficiently removing KRas4B from the membrane while SmgGDS-607 does not interfere with KRas4B membrane localization.

Aimed at reproducing the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely tethered bilayer lipid membranes, reveal the structural aspects of neurotransmitters’ association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts nonspecifically with the membrane at physiologically relevant concentrations, whereas γ-aminobutyric acid does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity, and neutron reflectometry shows that it penetrates the membrane deeply, whereas γ-aminobutyric is excluded from the bilayer.

Small lipidated GTPases such as Arf1 form dynamic signaling platforms with their regulators and effectors at the cell membrane surface to orchestrate key elements of cell membrane signaling and trafficking. However, there is incomplete knowledge as to how they interact with each other and with the membrane. Here, we focus on the interaction between Arf1 and the core catalytic domain of its Arf GTPase-activating protein ASAP1 (composed of PH, Arf GAP and Ankyrin repeat domains, PZA). Previous studies have shown that the Arf Gap activity of the PZA requires coincidence detection of the PH domain with Arf1 and multiple PIP2 headgroups at the membrane interface. The membrane docking geometry of the PH domain to PI(4,5)P2 containing bilayers was determined by combining constraints from (i) NMR PRE experiments of the 13C methyl labeled PH domain bound to nanodiscs (utilizing two types of data from either nanodiscs doped with doxyl-labeled lipids or solvent PRE in the presence of a paramagnetic caged compound); orientation and depth of insertion data from (ii) Neutron Reflectometry experiments and (iii) all atom MD simulations. Data show that the β1/β2 and β3β4 loops are involved in the membrane surface binding interface with PI(4,5)P2. The deepest protein backbone atom, Cα of residue I353 (β1/β2 loop), resides at the phosphate plane and its sidechain inserts below the average depths of headgroup phosphates. The long axis of the core β-sandwich lies at an angle 90o relative to the same plane. Examination of the PH-Arf1 reconstituted complex at the surface of a nanodisc shows that binding to multiple PIP2 orients the PH domain to facilitate association with switch 2 of Arf1.

Significance The proto-oncogene KRAS , a small GTPase, is frequently mutated in pancreatic, colorectal, and lung cancer. These mutations result in elevated levels of the activated guanosine triphosphate-bound form of KRAS. Localized at the plasma membrane, KRAS functions to recruit effectors, predominantly RAF kinase for activation and initiation of the MAPK signaling cascade. Combining computational and biophysical methods we identify a membrane-distal state of the KRAS G-domain that alternates with two previously described membrane-proximal states through dynamic reorganization of the hypervariable region. Comprising about 90% of the ensemble, this membrane-distal state of the G-domain dominates the proximal states and may facilitate KRAS to recruit cytosolic RAF kinase to the membrane by a fly-casting mechanism.

Due to their ubiquity, versatility, and chemical diversity, lipid bilayers are an increasingly important component of engineered biosensors and biophysical measurement platforms. Bilayers supported on solid substrates are a common design feature of these systems, and the presence of the substrate introduces perturbations in the bilayer structure, affecting both stability and function. Thus, the ability to understand and finely tune the physical interaction between a bilayer and a substrate is essential. Here we show that the separation between a bilayer and its supporting substrate can be finely tuned at the 0.1 nm level, as measured by neutron reflectivity, by adjusting the surface charge of the substrate. Remarkably, the tuning is possible even with zwitterionic bilayers, which appear to carry a small but non-negligible surface charge. The combination of van der Waals and electrostatic forces therefore appears to dominate the bilayer-substrate interaction. The interaction free energy profiles derived from a DLVO-type theory with no free parameters quantitatively describes the experimental observations.

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.

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.

Continuous solvent exchange has been shown to be a reliable and simple method to form supported planar lipid bilayers on solid substrates, particularly those that are not amenable to vesicle fusion. Here we show that bilayers formed in this way can be manipulated using the characteristic surface properties of these substrates. In addition, the mechanism of formation involves an initial partial monolayer of lipid near the substrate that serves as a template for bilayer assembly during the solvent exchange process. Using neutron reflectometry, we determine the structural features, especially the bilayer-substrate separation distance and the amplitude of bilayer fluctuations, of solid supported bilayers on TiOx and SiOx thin films. On TiOx films, the separation of a bilayer from the substrate is tunable from 1 to 5 nm by adjusting the pH and ionic strength of the bulk solvent. On the other hand, the separation distance of bilayers from SiOx control films shows no variation with pH. The difference can be attributed to the strong and weak variations, respectively, of the TiOx and SiOx surface charge over the pH range studied. A free energy model is developed to predict bilayer stability based on electrostatic contributions derived from double layer theory, van der Waals forces, steric repulsion, and short-range hydration forces. The model accurately predicts the observed bilayer-substrate separations and accounts for the increased bilayer fluctuations when the bilayer is far from the substrate. Implications for new measurement platforms and devices involving tunable floating bilayers are discussed.

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.

Human Immunodeficiency Virus-1 Nef is an accessory protein essential for the progression of HIV-1 infection by aiding viral production by down-regulating defense mechanisms of the infected cell to help evade immune response. Nef has been shown to interact with Src- and Tec-family kinases at the plasma membrane of infected cells, leading to constitutive kinase activation. For example, blocking Interleukin-2-inducable T-cell Kinase (Itk) activity with pharmacological inhibitors has been shown to result in decreased viral spread. Evidence also shows that Nef dimerizes on the membrane as it recruits kinases. However, the exact mechanism of this interaction between Nef and the kinases, or indeed between those two classes of proteins and the membrane, is not much understood. We used Surface Plasmon Resonance to study the binding of Nef on sparsely-tethered bilayer lipid membranes (stBLMs) in preparation for future studies in complex with kinases. Nef exhibits two distinct binding modes, one fast and one slow, which we attribute to two different membrane binding sites on Nef. The fast binding mode is likely that of the expected binding mechanism for Nef; insertion into the membrane of a myristoyl tail, whereby the core region of the protein is distant from the membrane surface. The second binding mode has Nef in much closer proximity to the membrane, with a much higher surface coverage. This in turn blocks the fast binding mode in subsequent concentration additions. For a given concentration, the binding rate is far higher when that concentration is added directly to a neat bilayer, rather than reached through a series of concentration additions. Previous Neutron Reflectometry (NR) characterization of membrane-bound Nef showed a concentration-dependent conformation change, consistent with this interpretation.

Neutron reflectometry (NR) is a powerful technique for probing the structure of lipid bilayer membranes and membrane-associated proteins. Measurements of the specular neutron reflectivity as a function of momentum transfer can be performed in aqueous environments, and inversion of the resulting reflectivity data yields structural profiles along the membrane normal with a spatial resolution approaching a fraction of a nanometer. With the inherent ability of the neutron to penetrate macroscopic distances through surrounding material, neutron reflectivity measurements provide unique structural information on biomimetic, fully hydrated model membranes and associated proteins under physiological conditions. A particular strength of NR is in the characterization of structurally and conformationally flexible peripheral membrane proteins. The unique ability of neutron scattering to differentiate protium from selectively substituted deuterium enables the resolution of individual constituents of membrane-bound protein-protein complexes. Integrative modeling strategies that supplement the low-resolution reflectometry data with complementary experimental and computational information yield high-resolution threedimensional models of membrane-bound protein structures.