The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins [1] into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (ld) lipid domains and recruits anionic lipids by an effective, electrostatic lipid sorting mechanism to form new protein-containing fluid domains with higher anionic charge density. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the ld phase and concentrate at the ld/lo phase boundary of heterogeneous membranes [2,3]. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.
478-Pos Board B278Elucidation of the Integrin Inside-Out Activation Mechanism Antreas Kalli, Iain D. Campbell, Mark S.P. Sansom. Integrins are major cell surface receptors that are crucial for a variety of cell migration and adhesion events. They are 'activated' to a high affinity state by the intracellular protein talin, a process known as ''inside-out activation''. In this process, complex formation between the talin head domain and the integrin b cytoplasmic tail, as well as talin/membrane interactions, are believed to play a crucial role. In this study, long multi-scale molecular dynamic simulations were used to probe the talin F2-F3/membrane and talin F3/b1D interactions in a POPC/POPG bilayer. A reorientation of the talin F2-F3 domain to optimize contacts with the negatively charged moieties in the membrane, followed by a large increase in the tilt angle of the b1D tail relative to the bilayer normal was observed. In addition, our simulations demonstrate that mutation of four basic residues in the F2 domain of talin, previously suggested to be involved in membrane interactions, reduces the affinity of talin F2-F3 for the membrane and changes its orientation relative to the bilayer surface. This perturbed orientation of talin relative to the membrane in the F2 mutant is expected, in turn, to perturb talin/integrin interactions. During the simulations, enrichment of the F2-F3 binding surface with anionic lipids reveals an important role for negatively...
By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.
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