Inhibition of K-RAS4B by a Unique Mechanism of Action: Stabilizing Membrane-Dependent Occlusion of the Effector-Binding Site

Fang, Zhenhao; Marshall, Christopher B.; Nishikawa, T.; Gossert, Alvar D.; Jansen, Johanna M.; Jahnke, Wolfgang; Ikura, Mitsuhiko

doi:10.1016/j.chembiol.2018.07.009

Cited by 81 publications

(96 citation statements)

References 33 publications

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“…Although it is long established that lipidated HVRs helps Ras GTPases anchor to the membrane, decades of research into the development of compounds that interfere with HVR lipidation, processing, and subsequent binding to membranes have failed to provide effective therapeutic reagents (70,71). Thus the binding interface between G-domain and membrane may provide a novel avenue to target K-Ras, as indeed has been very recently reported (63).…”

Section: K-ras Binding To Pip2mentioning

confidence: 55%

“…In a simulation study, Prakash et al (40) reported the involvement of helices 3 and 4, and ␤-strands 1-3, as well as helix 2 on the opposite face of the catalytic domain in the K-Ras4B G12D interaction with POPS/POPC bilayers. More recently, the interaction of C terminally membrane-anchored K-Ras4B with PS was also studied experimentally by Ikura and colleagues (44,63) using nanodiscs. This study revealed two, if not possibly more, orientation states, but also suggested a GTPase nucleotide and effector-bound state-specific shift between these states.…”

Section: Discussionmentioning

confidence: 99%

“…To our surprise, this protocol did not work well and a TLC assay showed that K-Ras WT remained bound to GDP, whereas G12V was initially bound to GTP, but hydrolyzed to GDP over several hours at room temperature. Thus, to obtain fully loaded K-Ras, the more elaborate loading protocol with a phosphatase to hydrolyze GTP and GDP is needed (63). The catalytic domain of p120 RasGAP (a gift from Dr. R. Ahmadian) was subcloned into pET28 and expressed and purified as described previously (94).…”

Section: K-ras Binding To Pip2mentioning

confidence: 99%

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K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

Cao

Chung

Kim

et al. 2019

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanRas genes potently drive human cancers, with mutated protooncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysicaland cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)phosphate (PIP2). We discovered that the ␤2 and ␤3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

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Section: K-ras Binding To Pip2mentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Section: K-ras Binding To Pip2mentioning

confidence: 99%

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K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

Cao

Chung

Kim

et al. 2019

Journal of Biological Chemistry

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“…5B, 5E-5G). Previously described exposed and occluded orientations (38)(39)(40) are included in the α and β/β' states, respectively, and an experimentally-driven model of RAS-RAF association straddles the dividing line between the α and β states (39). HMM analysis indicates that direct transitions between β' and α are relatively rare (Fig.…”

Section: Lipid Dependence Of Ras Orientation and Competence For Effecmentioning

confidence: 99%

Machine Learning-driven Multiscale Modeling Reveals Lipid-Dependent Dynamics of RAS Signaling Proteins

Ingólfsson¹,

Neale²,

Carpenter³

et al. 2020

Preprint

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RAS is a signaling protein associated with the cell membrane that is mutated in 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

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“…25 Their follow-up NMR study showed that this compound likely inhibits the function of K-Ras from the membrane surface. 26 Design of drugs that target the protein-membrane interactions is a relatively new idea. It this study, we propose a strategy of trapping K-Ras onto the membrane using membrane anchored cell penetrating peptides.…”

Section: Introductionmentioning

confidence: 99%

Computational Design of Myristoylated Cell Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector Binding Membrane Interface

Buck

2019

Preprint

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A number of small inhibitors have been developed in the recent years to target the cancer driving protein, K-Ras. In this study, we propose and design a novel way of targeting oncogenic K-Ras4B.G12D with myristoylated cell penetrating peptides which become membrane anchored and lock the protein into an inactive state. In all atom molecular dynamics simulations such peptides associate with K-Ras4B exclusively at the effector binding region, which, in turn, hinders the binding of down-stream effector proteins (e.g. C-Raf). The myristoylated R9 (Arg 9 ) peptide strongly locks K-Ras4B.G12D into orientations that are unfavorable for effector binding. After breaking the cyclic structure and myristoylation, a cell penetrating peptide cyclorasin 9A5, which was designed for targeting the Ras: Raf interface, is also found to be effective in targeting the Ras: membrane interface. The myristolyated peptides likely have high cell permeability due to their mixed cationic/hydrophobic character at the Nterminus, while simultaneously the subsequent multiple charges help to maintain a strong association of the peptide with the K-Ras4B.G12D effector binding lobe. Targeting protein-membrane interfaces is starting to attract attention very recently, thanks to our understanding of the signal mechanism of an increased number of peripheral membrane proteins. The strategy used in this study should have potential applications in the design of drugs against K-Ras4B driven cancers. It also provides insights into the general principles of targeting protein-membrane interfaces.

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Inhibition of K-RAS4B by a Unique Mechanism of Action: Stabilizing Membrane-Dependent Occlusion of the Effector-Binding Site

Cited by 81 publications

References 33 publications

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

Machine Learning-driven Multiscale Modeling Reveals Lipid-Dependent Dynamics of RAS Signaling Proteins

Computational Design of Myristoylated Cell Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector Binding Membrane Interface

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