| Autor: |
Prakash P; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Sayyed-Ahmad A; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Cho KJ; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Dolino DM; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Chen W; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Li H; University of Michigan Medical School, Department of Computational Medicine and Bioinformatics, Ann Arbor, Michigan, USA., Grant BJ; University of Michigan Medical School, Department of Computational Medicine and Bioinformatics, Ann Arbor, Michigan, USA., Hancock JF; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA., Gorfe AA; University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin St., Houston, Texas, 77030, USA. |
| Abstrakt: |
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. |
