Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers (original) (raw)

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes. Ras proteins are intracellular guanine tri-phosphate (GTP) hydrolyzing enzymes (GTPases) that mediate signal transduction from the extracellular environment to the nucleus 1,2. Signaling through Ras is achieved via a switch-like off/on conformational change driven by guanine di-phosphate (GDP) and GTP exchange. Malfunction in the switching function of the three human Ras isoforms N-, Hand K-Ras (4 A&B) due to somatic mutations is linked to 15-25% of all human cancers. Up to 85% of these are due to mutations in K-Ras4B (here after K-Ras), and include some of the most lethal cancers such as pancreatic and colorectal cancers 3. K-Ras interacts with effectors and exchange factors via a conserved catalytic domain comprising the first 166 of 185 residues, and with the plasma membrane (pm) via a farnesylated C-terminus carrying six lysines. The isolated catalytic domain as well as full-length K-Ras can bind effectors and hydrolyze GTP in their monomeric form 4,5. Previous studies thus largely focused on the monomer even though Ras dimerization has been proposed as far back as 1988 6 and 2000 7. This changed recently with the finding that dimers of endogenous K-Ras activate the MAPK pathway 8. There is evidence that Ras dimers also form in synthetic membranes. Gerwert and colleagues used fluorescence energy transfer (FRET) and Fourier transform infrared (FTIR) spectroscopies plus molecular dynamics (MD) simulation to propose that N-Ras forms dimer in a POPC bilayer 9. Similarly, using fluorescence correlation spectroscopy (FCS) and other techniques Groves and colleagues proposed dimerization of H-Ras in a supported bilayer 10 due to oxidative covalent interactions 11. The evidence for Ras dimerization in solution is mixed. For