Interaction of KRas4b with anionic membranes: A special role for PIP 2

Gregory, Michael C.; McLean, Mark A.; Sligar, Stephen G.

doi:10.1016/j.bbrc.2017.04.063

Cited by 49 publications

(79 citation statements)

References 25 publications

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“…This result directly links internal conformational fluctuation to membrane reorientation. We would like to emphasize that, although the different orientations are sampled spontaneously, they may be stabilized by protein-lipid electrostatic interactions as suggested by several previous reports (16,18,23). That intrinsic flexibility underlies the membrane orientation dynamics of G12V-KRAS is further confirmed by performing another 20 ms MD simulation using the CHARMM36 force field (24).…”

Section: G12v-kras Adopts Three Distinct Orientations With Respect Tosupporting

confidence: 68%

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Prakash

Litwin

Luan

et al. 2019

Biophysical Journal

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Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal b-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 ms-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.

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Section: G12v-kras Adopts Three Distinct Orientations With Respect Tosupporting

confidence: 68%

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Prakash

Litwin

Luan

et al. 2019

Biophysical Journal

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“…The results show that the two orientations have remained stable over the course of each of the trajectories. It should be noted that for our previous K-Ras4A [Li et al, 2017] and K-Ras4B [Gregory et al, 2017] simulations with PIP2, primarily the OS1 state was found to be populated (similar to O3 in Li et al, 2017). The OS2 started simulation may not be able to interconvert to OS1 on the timescale of the current simulations, also having been stabilized by PIP2 contacts; such transition was seen, however, by Gregory et al using a membrane model with increased fluidity.…”

Section: K-ras4b On Pi(45)p2 Membrane -Interface and Orientations Stcontrasting

confidence: 51%

K-Ras G-domain binding with signaling lipid phosphoinositides: PIP2 association, orientation, function

Cao

Chung

Kim

et al. 2018

Preprint

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Ras genes are potent drivers of human cancers, with mutated 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 inhibitors for KRas4B greatly benefited from a deeper understanding of the protein structure and dynamics of the GTPase. Since interactions with biological membranes are key for Ras function, the details of Ras -lipid interactions have become a major focus of study, especially since it is becoming clear that such interactions not only involve the Ras C-terminus for lipid anchoring, but also the G-protein domain. Here we investigated the interaction between K-Ras4B with the signaling lipid phosphatidyl inositol (4,5) phosphate (PIP2) using NMR spectroscopy and molecular dynamics simulations, complemented by biophysical and cell biology assays. We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2, and that these secondary structural elements employ specific residues for these interactions. These likely occur in two orientation states of the protein relative to the membrane. Importantly, we found that some of these residues, which are known to be oncogenic when mutated (D47K, D92N, K104M and D126N), are critical for K-Ras-mediated transformation of fibroblast cells, while not substantially affecting basal and assisted nucleotide hydrolysis and exchange. We further showed that mutation K104M can indeed abolish localization of mutant K-Ras to the plasma membrane. These findings suggest that specific G-domain residues play an important, previously-unknown role in regulating Ras function by mediating interactions with membrane PIP2 lipids. Thus, a detailed description of the novel K-Ras-PIP2 binding surfaces is likely to inform the future design of therapeutic reagents.

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“…It should be noted that for our previous K-Ras4A (42) and K-Ras4B (40) simulations with PIP2, primarily the OS1 state was found to be populated (similar to O3 in Li and Buck (42)). The OS2 started simulation may not be able to interconvert to OS1 on the timescale of the current simulations, also having been stabilized by PIP2 contacts; such transition was seen, however, by Gregory et al (43) using a membrane model with increased fluidity.…”

Section: K-ras4b On Pi(45)p2 Membrane-interface and Orientations Stumentioning

confidence: 65%

“…G-domain orientation) and dynamics at the membrane. For example, recent computational investigations suggested the participation of the K-Ras.G12V G-domain in binding with POPS membrane (39 -42) and with PIP2 containing membranes (42,43). Experimental work by Ikura and colleagues (44) also provided evidence that the K-Ras G-domain exists in several distinct orientations when contacting a bilayer of POPS lipid membrane.…”

mentioning

confidence: 99%

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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Interaction of KRas4b with anionic membranes: A special role for PIP 2

Cited by 49 publications

References 25 publications

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

K-Ras G-domain binding with signaling lipid phosphoinositides: PIP2 association, orientation, function

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

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