KRas4b is a key drivers of signal transduction events that control cell division, proliferation, and apoptosis. KRas4b activity is controlled, and signaling mediated by, critical protein-protein interactions with GTPase activating proteins (GAPs), and guanine nucleotide exchange factors (GEFs).  Importantly, these protein complexes are located on the membrane surface where membrane-protein interactions play a key role.  However, there is incomplete knowledge as to the role of the bilayer in regards to membrane binding, the importance of specific lipid type in dictating the affinity and orientation of KRas4b on the surface and the dynamics of this important membrane-protein interface.  We have approached this problem experimentally by using a fully post-translationally modified Kras4b provided by the Frederick National Laboratory and the Nanodisc system, which allows precise control of surface area and lipid identity.  We experimentally investigated the role of specific lipids in the recruitment of KRas4b to the Nanodisc membrane surface by monitoring the rotational diffusion of the KRas4b-Nanodisc system in solution using multi-frequency (100 kHz-10 MHz) phase fluorometry and orientation by slab waveguide linear dichroism. However, understanding the molecular origins of the observed binding specificity as well as the topology of KRas4b on the surface requires long-time molecular dynamics simulation.  Through extensive (multiple microsecond) trajectories it appears that the significant binding preference for Nanodisc bilayers containing the polyvalent PIP2 and PIP3 results from long lived salt bridges formed between the positively charged residues in the Hyper Variable Region (HVR).  Interestingly, long-lived membrane interactions also occur on the globular domain, and appear to control the orientation of KRas4b at the membrane surface.  For instance, Helix-4 is seen to interact with PIP2 though a bifurcated salt bridge between Arg 135 and Lys 128 which leads to an orientation of Kras4b poised for interaction with effector proteins.  Mutations of Arg 135 and Lys 128 to Ala do not dramatically alter the affinity for PIP2 membranes but significantly affect the conformational equilibrium as probed by molecular dynamics simulations. Our results show that the membrane itself plays an important role in localizing signaling proteins and in the control of critical molecular recognition events.<br/>

Proteins interacting with membrane surfaces: The case of the oncogene KRas4b

2019 Spring National Meeting

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