From disorder comes function: Regulation of small GTPase function by intrinsically disordered lipidated membrane anchor.

The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs).

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Intrinsically disordered membrane anchors of Rheb, RhoA and DiRas3 small GTPases: Molecular dynamics, membrane organization, and interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell signaling processes. Nearly all Ras Superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of these IDRs are key determinants of membrane localization, and interactions between the IDR and the membrane have been shown to affect signaling in RAS proteins through modulation of dynamic membrane organization. Here we utilized atomistic molecular dynamics simulations to study the membrane interactions, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs generate semi-stable conformational sub-states, and a lack of these residues leads to greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic membrane lipids, and, in some cases, excluding other lipids from their immediate proximity in favor of anionic lipids.

Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases.

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function.

DIRAS3 is an imprinted tumor suppressor gene encoding a GTPase that has a distinctive N-terminal extension (NTE) not found in other RAS proteins. This NTE and the prenylated C-terminus are required for DIRAS3-mediated inhibition of RAS/MAP signaling and PI3K activity at the plasma membrane. In this study, we applied biochemical, biophysical, and computational methods to characterize the structure and function of the NTE. The NTE peptide recognizes phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 with rapid kinetics and strong affinity. Lipid binding induces NTE structural change from disorder to amphipathic helix. Mass spectrometry identified N-myristoylation of DIRAS3. All-atom molecular dynamic simulations predict DIRAS3 could adhere to the membrane through both termini, suggesting the NTE is involved in targeting and stabilizing DIRAS3 on the membrane by double anchoring. Overall, our results are consistent with DIRAS3's function as a tumor suppressor, whereby the membrane-bound DIRAS3 can effectively target PI3K and KRAS at the membrane.

A new ferrocene derivative blocks K-Ras localization and function by oxidative modification at His95.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers harboring oncogenic mutant K-Ras. Here, we tested a novel ferrocene derivative on the growth of pancreatic ductal adenocarcinoma and non-small cell lung cancer. Our compound, which elevated cellular ROS levels, inhibited the growth of K-Ras-driven cancers, and abrogated the PM binding and signaling of K-Ras in an isoform-specific manner. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified that K-Ras His95 residue plays an important role in this process, and it is putatively oxidized by cellular ROS. Together, our study demonstrates that the redox system directly regulates K-Ras/PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced growth and metastasis of K-Ras-driven cancers.

Small-Molecule Inhibition of KRAS through Conformational Selection.

Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] binds to KRAS with low micro-molar affinity [Chem. Biol. Drug Des.2019; 94(2):1441-1456]. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.

Conformational ensemble dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases.

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. We show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we found that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

A new ferrocene derivative blocks KRAS localization and function by oxidative modification at His95.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers.

"Quality teaches you how to use water. It doesn't provide a water pump": a qualitative study of context and mechanisms of action in an Ethiopian quality improvement program.

BACKGROUND: Quality improvement collaboratives are a common approach to bridging the quality-of-care gap, but little is known about implementation in low-income settings. Implementers rarely consider mechanisms of change or the role of context, which may explain collaboratives' varied impacts. METHODS: To understand mechanisms and contextual influences we conducted 55 in-depth interviews with staff from four health centres and two hospitals involved in quality improvement collaboratives in Ethiopia. We also generated control charts for selected indicators to explore any impacts of the collaboratives. RESULTS: The cross facility learning sessions increased the prominence and focus on quality, allowed learning from experts and peers and were motivational through public recognition of success or a desire to emulate peers. Within facilities, new structures and processes were created. These were fragile and sometimes alienating to those outside the improvement team. The trusted and respected mentors were important for support, motivation and accountability. Where mentor visits were infrequent or mentors less skilled, team function was impacted. These mechanisms were more prominent, and quality improvement more functional, in facilities with strong leadership and pre-existing good teamwork; as staff had shared goals, an active approach to problems and were more willing and able to be flexible to implement change ideas. Quality improvement structures and processes were more likely to be internally driven and knowledge transferred to other staff in these facilities, which reduced the impact of staff turnover and increased buy-in. In facilities which lacked essential inputs, staff struggled to see how the collaborative could meaningfully improve quality and were less likely to have functioning quality improvement. The unexpected civil unrest in one region strongly disrupted the health system and the collaborative. These contextual issues were fluid, with multiple interactions and linkages. CONCLUSIONS: The study confirms the need to carefully consider context in the implementation of quality improvement collaboratives. Facilities that implement quality improvement successfully may be those that already have characteristics that foster quality. Quality improvement may be alienating to those outside of the improvement team and implementers should not assume the organic spread or transfer of quality improvement knowledge.

Remodeling of the Plasma Membrane by Surface-Bound Protein Monomers and Oligomers: The Critical Role of Intrinsically Disordered Regions.

The plasma membrane (PM) of cells is a dynamic structure whose morphology and composition is in constant flux. PM morphologic changes are particularly relevant for the assembly and disassembly of signaling platforms involving surface-bound signaling proteins, as well as for many other mechanochemical processes that occur at the PM surface. Surface-bound membrane proteins (SBMP) require efficient association with the PM for their function, which is often achieved by the coordinated interactions of intrinsically disordered regions (IDRs) and globular domains with membrane lipids. This review focuses on the role of IDR-containing SBMPs in remodeling the composition and curvature of the PM. The ability of IDR-bearing SBMPs to remodel the Gaussian and mean curvature energies of the PM is intimately linked to their ability to sort subsets of phospholipids into nanoclusters. We therefore discuss how IDRs of many SBMPs encode lipid-binding specificity or facilitate cluster formation, both of which increase their membrane remodeling capacity, and how SBMP oligomers alter membrane shape by monolayer surface area expansion and molecular crowding.

Phosphatidylserine and Phosphatidylethanolamine Asymmetry Have a Negligible Effect on the Global Structure, Dynamics, and Interactions of the KRAS Lipid Anchor.

The intrinsically disordered C-terminus of the prominent oncogenic protein KRAS-4B (KRAS) selectively interacts and clusters with phosphatidylserine (PS) lipids in the plasma membrane (PM). This 11-residue segment, called tK, contains a polybasic domain (PBD) of six contiguous lysine residues and a farnesylated cysteine. Previous molecular dynamics (MD) simulation studies of tK in phosphatidylcholine (PC)/PS bilayers have suggested that backbone conformational dynamics modulate tK-PS interactions. These simulations have been conducted in symmetric membranes whereas the PM is compositionally asymmetric, with the inner leaflet, where KRAS is localized, being enriched with PS and phosphatidylethanolamine (PE) lipids. To examine if bilayer asymmetry affects tK conformational dynamics and interaction with lipids, we conducted two 10 µs long MD simulations of tK bound to a PC/PS and a PC/PS/PE bilayer in which the PS and PE lipids are distributed in one leaflet. We found that, first, these compositional asymmetries caused differences in acyl chain dynamics between leaflets, but the equilibrium structural and dynamic properties of the two asymmetric bilayers are similar; second, in both systems tK is highly dynamic and samples at least two distinct conformational states; third, PS-tK hydrogen-bonding interactions vary with peptide backbone conformations, and lysine side chains in the PBD predominantly interact with the serine oxygens of PS. These results are in good agreement with previous observations of tK in symmetric membranes. The effects of POPS asymmetry or the presence of POPE on tK are limited to modulating the relative contribution of individual side chains to interactions with lipids and redistributing conformational substates. Additional observations include the larger flexibility of tK in the current simulations, which we attribute to the longer duration of the simulations and the use of the CHARMM36m force field, which more accurately models intrinsically disordered peptides such as tK.

Determinants of Membrane Orientation Dynamics in Lipid-Modified Small GTPases.

The transient membrane engagement and reorientation of the soluble catalytic domain of Ras proteins has emerged as an important modulator of their functions. However, there has been limited information on whether this phenomenon is applicable to other members of the Ras superfamily. To address this issue, we conducted long-time-scale atomistic molecular dynamics simulations (55 µs aggregate simulation time) on representatives of the Ras, Rho, and Arf family proteins that differ in sequence, lipid modification, and the rigidity of the linker between the lipid anchor and the catalytic G-domain. The results show that the concept of membrane reorientation is generalizable to most but not all members of the Ras superfamily. Specifically, C-terminally prenylated small GTPases that are anchored to membranes via a single flexible linker adopt multiple orientations, whereas those that are N-terminally myristoylated and harbor a rigid linker experience limited orientational dynamics. Combined with published reports on Ras proteins, these observations provide insights into the common principles and determinants of the orientational dynamics of lipidated small GTPases on membrane surfaces and offer new ways of thinking about the regulation and druggability of the Ras superfamily proteins.

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

RAS Nanoclusters Selectively Sort Distinct Lipid Headgroups and Acyl Chains.

RAS proteins are lipid-anchored small GTPases that switch between the GTP-bound active and GDP-bound inactive states. RAS isoforms, including HRAS, NRAS and splice variants KRAS4A and KRAS4B, are some of the most frequently mutated proteins in cancer. In particular, constitutively active mutants of KRAS comprise ∼80% of all RAS oncogenic mutations and are found in 98% of pancreatic, 45% of colorectal and 31% of lung tumors. Plasma membrane (PM) is the primary location of RAS signaling in biology and pathology. Thus, a better understanding of how RAS proteins localize to and distribute on the PM is critical to better comprehend RAS biology and to develop new strategies to treat RAS pathology. In this review, we discuss recent findings on how RAS proteins sort lipids as they undergo macromolecular assembly on the PM. We also discuss how RAS/lipid nanoclusters serve as signaling platforms for the efficient recruitment of effectors and signal transduction, and how perturbing the PM biophysical properties affect the spatial distribution of RAS isoforms and their functions.

Molecular Dynamics Simulation of Lipid-Modified Signaling Proteins.

In this chapter, we provide a practical guide on how to plan, execute, and interpret atomistic and coarse-grained molecular dynamics (MD) simulations of lipid-modified proteins in model membranes. After outlining some key practical considerations when planning such simulations, we survey resources and techniques to obtain force field parameters for nonconventional amino acids, such as posttranslationally lipid-modified amino acids that are unique to this class of proteins. We then describe the protocols to build, setup, and run the simulations, followed by a brief comment on the analysis and interpretation of the simulations. Finally, examples of insights that could be gained from atomistic and coarse-grained MD simulations of lipidated proteins will be provided, using RAS proteins as illustrative examples. Throughout the chapter, we highlight the main advantages and limitations of simulating RAS and related lipid-modified G-proteins in biomimetic membranes.

Antipsychotic phenothiazine drugs bind to KRAS in vitro.

We used NMR to show that the antipsychotic phenothiazine drugs promazine and promethazine bind to GDP-KRAS. Promazine also binds to oncogenic GDP-KRAS(G12D), and to wild type GppNHp-KRAS. A panel of additional phenothiazines bind to GDP-KRAS but with lower affinity than promazine or promethazine. Binding is most dependent on substitutions at C-2 of the tricyclic phenothiazine ring. Promazine was used to generate an NMR-driven HADDOCK model of the drug/GDP-KRAS complex. The structural model shows the tricyclic phenothiazine ring of promazine associates with the hydrophobic pocket p1 that is bordered by the central ß sheet and Switch II in KRAS. Binding appears to stabilize helix 2 in a conformation that is similar to that seen in KRAS bound to other small molecules. Association of phenothiazines with KRAS may affect normal KRAS signaling that could contribute to multiple biological activities of these antipsychotic drugs. Moreover, the phenothiazine ring represents a new core scaffold on which to design modulators of KRAS activity.