Abstract

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.

Highlights

  • Recent studies found that membrane-bound K-Ras dimers are important for biological function

  • Signaling through Ras is achieved via a switch-like off/on conformational change driven by guanine di-phosphate (GDP) and guanine tri-phosphate (GTP) exchange

  • 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]

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Summary

Introduction

Recent studies found that membrane-bound K-Ras dimers are important for biological function. We show that the catalytic domain of K-Ras is directly involved in the formation of multiple dimers, but the PPIs are so weak that dimers are unlikely to be observed under standard experimental conditions in solution but can be enriched upon membrane binding.

Results
Conclusion

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