Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

Que N. Van; Cesar A. López; Marco Tonelli; Troy Taylor; Ben Niu; Christopher B. Stanley; Debsindhu Bhowmik; Timothy H. Tran; Peter H. Frank; Simon Messing; Patrick Alexander; Daniel Scott; Xiaoying Ye; Matt Drew; Oleg Chertov; Mathias Lösche; Arvind Ramanathan; Michael L. Gross; Nicolas W. Hengartner; William M. Westler; John L. Markley; Dhirendra K. Simanshu; Dwight V. Nissley; William K. Gillette; Dominic Esposito; Frank McCormick; S. Gnanakaran; Frank Heinrich; Andrew G. Stephen

doi:10.1073/pnas.2006504117

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

Que N. Van, Cesar A. López, Marco Tonelli, Troy Taylor, Ben Niu, Christopher B. Stanley, Debsindhu Bhowmik, Timothy H. Tran, Peter H. Frank, Simon Messing, Patrick Alexander, Daniel Scott, Xiaoying Ye, Matt Drew, Oleg Chertov, Mathias Lösche, Arvind Ramanathan, Michael L. Gross, Nicolas W. Hengartner, William M. WestlerJohn L. Markley, Dhirendra K. Simanshu, Dwight V. Nissley, William K. Gillette, Dominic Esposito, Frank McCormick, S. Gnanakaran, Frank Heinrich, Andrew G. Stephen

Biostatistics and Biomedical Informatics

Research output: Contribution to journal › Article › peer-review

38 Scopus citations

Abstract

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.

Original language	English
Pages (from-to)	24258-24268
Number of pages	11
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	117
Issue number	39
DOIs	https://doi.org/10.1073/pnas.2006504117
State	Published - Sep 29 2020

Funding

Q.N.V. thanks Amanda Altieri and Mike Gregory for sharing their nanodisc-making expertise and Donghan Lee for providing the Excel worksheet to calculate τc from TRACT data. We thank members of the Frederick National Laboratory for Cancer Research (FNLCR) Protein Expression Laboratory for help with cloning (Carissa Grose, Jennifer Mehalko, and Vanessa Wall), Escherichia coli expression (John-Paul Denson), insect expression (Kelly Snead), and protein purification (John-Paul Denson, Shelley Perkins, and Mukul Sherekar). We thank Timothy Waybright, FNLCR, for validation of GppNHp exchange using high-pressure liquid chromatography analysis. Computing resources were made available by Los Alamos National Laboratory Institutional Computing. Research was performed in part at the National Institute of Standards and Technology (NIST) Center for Nanoscale Science and Technology. Certain commercial materials, equipment, and instruments are identified in this work to describe the experimental procedure as completely as possible. In no case does such an identification imply a recommendation or endorsement by NIST, nor does it imply that the materials, equipment, or instrument identified are necessarily the best available for the purpose. This project was funded in whole or in part with federal funds from National Cancer Institute (NCI), NIH Contract HHSN261200800001E and the Spatiotemporal Modeling Center at the University of New Mexico (NIH P50GM085273). This study made use of the National Magnetic Resonance Facility at Madison, WI, which is supported by NIH grant P41GM103399. Equipment was purchased with funds from the University of Wisconsin-Madison, the NIH (P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, and S10RR029220), and the NSF (DMB-8415048, OIA-9977486, and BIR-9214394). The mass spectrometry-based footprinting was conducted at the NIH facility at Washington University in St. Louis (Grant p41GM103422). This work has been supported in part by the Joint Design of Advanced Computing Solutions for Cancer (JDACS4C) program established by the US Department of Energy (DOE) and the NCI of the NIH. This work was performed under the auspices of the US DOE by Argonne National Laboratory under Contract DE-AC02-06-CH11357, Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, Los Alamos National Laboratory under Contract DE-AC5206NA25396, Oak Ridge National Laboratory under Contract DE-AC05-00OR22725, and FNLCR under contract HHSN261200800001E. This work was supported by the US Department of Commerce (Award 70NANB17H299). ACKNOWLEDGMENTS. Q.N.V. thanks Amanda Altieri and Mike Gregory for sharing their nanodisc-making expertise and Donghan Lee for providing the Excel worksheet to calculate τc from TRACT data. We thank members of the Frederick National Laboratory for Cancer Research (FNLCR) Protein Expression Laboratory for help with cloning (Carissa Grose, Jennifer Mehalko, and Vanessa Wall), Escherichia coli expression (John-Paul Denson), insect expression (Kelly Snead), and protein purification (John-Paul Denson, Shelley Perkins, and Mukul Sherekar). We thank Timothy Waybright, FNLCR, for validation of GppNHp exchange using high-pressure liquid chromatography analysis. Computing resources were made available by Los Alamos National Laboratory Institutional Computing. Research was performed in part at the National Institute of Standards and Technology (NIST) Center for Nanoscale Science and Technology. Certain commercial materials, equipment, and instruments are identified in this work to describe the experimental procedure as completely as possible. In no case does such an identification imply a recommendation or endorsement by NIST, nor does it imply that the materials, equipment, or instrument identified are necessarily the best available for the purpose. This project was funded in whole or in part with federal funds from National Cancer Institute (NCI), NIH Contract HHSN261200800001E and the Spatiotemporal Modeling Center at the University of New Mexico (NIH P50GM085273). This study made use of the National Magnetic Resonance Facility at Madison, WI, which is supported by NIH grant P41GM103399. Equipment was purchased with funds from the University of Wisconsin–Madison, the NIH (P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, and S10RR029220), and the NSF (DMB-8415048, OIA-9977486, and BIR-9214394). The mass spectrometry-based footprinting was conducted at the NIH facility at Washington University in St. Louis (Grant p41GM103422). This work has been supported in part by the Joint Design of Advanced Computing Solutions for Cancer (JDACS4C) program established by the US Department of Energy (DOE) and the NCI of the NIH. This work was performed under the auspices of the US DOE by Argonne National Laboratory under Contract DE-AC02-06-CH11357, Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, Los Alamos National Laboratory under Contract DE-AC5206NA25396, Oak Ridge National Laboratory under Contract DE-AC05-00OR22725, and FNLCR under contract HHSN261200800001E. This work was supported by the US Department of Commerce (Award 70NANB17H299).

Funders	Funder number
Timothy Waybright
National Science Foundation	DMB-8415048, BIR-9214394, p41GM103422, OIA-9977486
National Science Foundation
National Institutes of Health	HHSN261200800001E
National Institutes of Health
U.S. Department of Energy
National Cancer Institute
National Center for Research Resources	S10RR025062
National Center for Research Resources
National Institute of Standards and Technology
U.S. Department of Commerce	70NANB17H299
U.S. Department of Commerce
Argonne National Laboratory	DE-AC02-06-CH11357
Argonne National Laboratory
Lawrence Livermore National Laboratory	DE-AC5206NA25396, DE-AC52-07NA27344
Lawrence Livermore National Laboratory
Oak Ridge National Laboratory	DE-AC05-00OR22725
Oak Ridge National Laboratory
University of Wisconsin-Madison	S10RR023438, S10RR08438, S10RR02781, S10RR029220
University of Wisconsin-Madison
University of New Mexico	P50GM085273, P41GM103399
University of New Mexico
Los Alamos National Laboratory
Frederick National Laboratory for Cancer Research

Keywords

KRAS
Membrane
Neutron reflectometry
Nuclear magnetic resonance
RAF RBD

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10.1073/pnas.2006504117

Cite this

Van, Q. N., López, C. A., Tonelli, M., Taylor, T., Niu, B., Stanley, C. B., Bhowmik, D., Tran, T. H., Frank, P. H., Messing, S., Alexander, P., Scott, D., Ye, X., Drew, M., Chertov, O., Lösche, M., Ramanathan, A., Gross, M. L., Hengartner, N. W., ... Stephen, A. G. (2020). Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane. Proceedings of the National Academy of Sciences of the United States of America, 117(39), 24258-24268. https://doi.org/10.1073/pnas.2006504117

@article{d770f4d3861249dba1949e0afce7ba3d,

title = "Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane",

abstract = "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.",

keywords = "KRAS, Membrane, Neutron reflectometry, Nuclear magnetic resonance, RAF RBD",

author = "Van, {Que N.} and L{\'o}pez, {Cesar A.} and Marco Tonelli and Troy Taylor and Ben Niu and Stanley, {Christopher B.} and Debsindhu Bhowmik and Tran, {Timothy H.} and Frank, {Peter H.} and Simon Messing and Patrick Alexander and Daniel Scott and Xiaoying Ye and Matt Drew and Oleg Chertov and Mathias L{\"o}sche and Arvind Ramanathan and Gross, {Michael L.} and Hengartner, {Nicolas W.} and Westler, {William M.} and Markley, {John L.} and Simanshu, {Dhirendra K.} and Nissley, {Dwight V.} and Gillette, {William K.} and Dominic Esposito and Frank McCormick and S. Gnanakaran and Frank Heinrich and Stephen, {Andrew G.}",

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doi = "10.1073/pnas.2006504117",

language = "English",

volume = "117",

pages = "24258--24268",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

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Van, QN, López, CA, Tonelli, M, Taylor, T, Niu, B, Stanley, CB , Bhowmik, D, Tran, TH, Frank, PH, Messing, S, Alexander, P, Scott, D, Ye, X, Drew, M, Chertov, O, Lösche, M, Ramanathan, A, Gross, ML, Hengartner, NW, Westler, WM, Markley, JL, Simanshu, DK, Nissley, DV, Gillette, WK, Esposito, D, McCormick, F, Gnanakaran, S, Heinrich, F & Stephen, AG 2020, 'Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane', Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 39, pp. 24258-24268. https://doi.org/10.1073/pnas.2006504117

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T1 - Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

AU - Van, Que N.

AU - López, Cesar A.

AU - Tonelli, Marco

AU - Taylor, Troy

AU - Niu, Ben

AU - Stanley, Christopher B.

AU - Bhowmik, Debsindhu

AU - Tran, Timothy H.

AU - Frank, Peter H.

AU - Messing, Simon

AU - Alexander, Patrick

AU - Scott, Daniel

AU - Ye, Xiaoying

AU - Drew, Matt

AU - Chertov, Oleg

AU - Lösche, Mathias

AU - Ramanathan, Arvind

AU - Gross, Michael L.

AU - Hengartner, Nicolas W.

AU - Westler, William M.

AU - Markley, John L.

AU - Simanshu, Dhirendra K.

AU - Nissley, Dwight V.

AU - Gillette, William K.

AU - Esposito, Dominic

AU - McCormick, Frank

AU - Gnanakaran, S.

AU - Heinrich, Frank

AU - Stephen, Andrew G.

PY - 2020/9/29

Y1 - 2020/9/29

N2 - 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.

AB - 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.

KW - KRAS

KW - Membrane

KW - Neutron reflectometry

KW - Nuclear magnetic resonance

KW - RAF RBD

UR - http://www.scopus.com/inward/record.url?scp=85092270731&partnerID=8YFLogxK

U2 - 10.1073/pnas.2006504117

DO - 10.1073/pnas.2006504117

M3 - Article

C2 - 32913056

AN - SCOPUS:85092270731

SN - 0027-8424

VL - 117

SP - 24258

EP - 24268

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 39

ER -

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

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