DEPARTMENT OF HEALTH AND HUMAN SERVICES • National Institutes of Health • National Cancer InstituteThe Frederick National Laboratory is a Federally Funded Research and Development Center operated by Leidos Biomedical Research, Inc., for the National Cancer Institute

Pilot 2 Introduction: Frank McCormick

?

RAS.GTP

RBD

CRD

Kinase domain

?

RAS.GTP recruits RAF to the plasma membrane

Plasmamembrane

Mek

RAS.GTP

Dimerized Active Kinase domain

?

?

RAS.GTP

CRD

RBDRBD

CRD?

Raf dimerization and activation

A multi-pronged approach to KRAS-RAF membrane interactions

In vivo(FNL and collaborators)

In vitro(FNL and collaborators)

In silico (DOE)

5

Frantz Jean-FrancoisAndy StephenTommy Turbyville

KRAS4b mobility characterized by three states in live cells

Dataset: RASless-MEF, HaloTag-KRAS4b; # Tracks: 29, 165; # Cells: 12; Avg. Track Length = 13.1 frames (frame = 10 ms)

2 4 6 88

9

10

11

12

13

14

15

X position [m]

Y po

sitio

n [

m]

Component Diffusion(um2/s) Fraction

Fast 0.97 48%

Intermediate 0.22 26%

Slow 0.039 26%

Slow

FastIntermediate

Accurate determination of binding constants

0 500 1000

0

500

1000

KRAS 4b FMe

Time (s)

Liposome 4

Liposome 5

Liposome 6

0 2 10 -06 4 10 -06 6 10 -06 8 10 -06 1 10 -050

200

400

600

800

1000

Liposome 5

[KRAS FMe](M)0 2 10 -06 4 10 -06 6 10 -06 8 10 -06 1 10 -05

0

200

400

600

800

1000

Liposome 6

[KRAS FMe](M)

KD=570 nM KD=700 nM KD=327 nM

10uM3.3uM

0.36uM

0.12uM

1.1uM

KRAS:membrane – structure and orientation

• KRAS4b protein shape and orientation on the membrane– Neutron reflectivity (National Institute of Standards and Technology)

– Small Angle Neutron/X-ray Scattering (Oak Ridge National Laboratory)

• Identify KRAS4b:membrane interacting residues– Protein foot printing (Washington University in St. Louis)

– NMR (National Magnetic Resonance Facility at Madison)

• Develop a model of KRAS on a membrane (Department of Energy)

Molecular dynamics

simulations

SAXS/SANS

NMR

NR

Protein foot printing

SAXS and SANS indicate KRAS exists in an extended conformation

9

~70

~80

• Ensemble MD simulations agree qualitatively with SANS/SAXS

Scattering Angle Q (Å-1) Scattering Angle Q (Å-1)

SANS SAXS

Chris Stanley, Sindhu Bhowmik, Blake Wilson, Arvind Ramanathan (ORNL)

Beta strand 1-3 and helix 2-5 are close to the membrane

Biophysical data indicates KRAS exists in an extended conformation at the membrane

farn

esyl

KKKKKSKTKC-Me

farn

esyl

N-term

N-termKMSKDGKKKKKKSKTKC-Me

~70 Å• KRAS is

predominantly extended away from the membrane.

• Orientation independent of nucleotide state

• N-terminal beta strands 1-3 and regions of helix 2-5 are proximal to the membrane