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Biochemistry Seminar: Dr. Andrew Stephen (Frederick National Laboratory for Cancer Research), "Ras Signaling in Human Cancer"

Event Type
academic, biochemistry
Dr. Stephen Sligar
Charles G. Miller Auditorium (B102 CLSL)
Nov 9, 2018   12:00 pm  
Dr. Andrew Stephen
Sherry Unkraut
Originating Calendar
Biochemistry Department Seminars

30% of all human cancers have RAS mutations. RAS is a small GTPase that cycles between an active GTP-bound state and inactive GDP-bound state. RAS exists as 4 isoforms with a highly conserved G-domain and variable C-terminus that traffics the protein to the plasma membrane. Oncogenic mutations lock RAS into the active GTP state. RAS-GTP binds to its primary effector RAF kinase at the plasma membrane, facilitating dimerization and initiative of the MAPK signaling cascade. The precise molecular details of this process are currently unknown. However, defining the structure of RAS complexed to RAF kinase at the membrane and mechanistic details of the activation process may provide novel strategies towards modulation of this interaction. We have used a variety of biophysical approaches to investigate the structural orientation of farnesylated and methylated KRAS4b (KRAS-FMe) alone or in complex with domains of RAF1 kinase in the presence of membrane mimetics. Using a combination of neutron reflectivity, NMR and protein footprinting we identified specific interactions between the KRAS-FMe HVR and the lipid bilayer. However no direct contacts between the G-domain and the bilayer were observed. The role of the HVR residues in direct membrane interaction was validated by mutational analysis. Analysis of NMR and neutron reflectivity data of the Ras Binding Domain (RBD) of RAF1 complexed with KRAS-FMe on membrane mimetics indicates that the binding of RBD does not significantly alter the orientation of KRAS on the membrane nor does RBD interact strongly with the membrane. Loops L62-R67 and L102-L112 and the beta strand at the C-terminus of RBD were oriented towards the bilayer when complexed with KRAS-FMe. Neutron reflectivity studies of KRAS-FMe bound with RBD on tethered lipid bilayers are consistent with the NMR measurements. NMR experiments with Cysteine Rich Domain from RAF1 indicates loops T145-F151 and F158-G162 interact directly with the bilayer of nanodiscs. Finally, the protein envelope obtained from neutron reflectivity measurements of KRAS-FMe:RBD-CRD on tethered lipid bilayers, is used to generate a model of the KRAS-FMe:RBD-CRD complex bound at the membrane.

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