Interaction of KRas4b with anionic membranes: A special role for PIP₂

KRas4b is a small G-protein whose constitutively active oncogenic mutants are present in 90% of pancreatic cancers. Using fully post-translationally modified KRAS4b, we investigated the role of lipid identity in the recruitment of KRas4b to a membrane surface of defined composition. Application of a newly developed single frequency fluorescence anisotropy decay experiment to this system revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP₂. We conducted molecular dynamics simulations to look for an origin of this specificity. In the case of membranes containing PIP₂ the protein formed long-lived salt bridges with PIP₂ head groups but not the monovalent DMPS, explaining the experimentally observed lipid specificity. Additionally, we report that PIP₂ forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient the protein in a manner expected to facilitate association with upstream and downstream signaling partners.