Mapping the Nucleotide and Isoform-Dependent Structural and Dynamical Features of Ras Proteins

149

165

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K d is measured to be ∼1 × 10 3 molecules/μm 2 , and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.Ras signaling | Ras assay

mentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

H-Ras forms dimers on membrane surfaces via a protein–protein interface

Lin

Iversen

et al. 2014

149

165

“…2. Pocket 1 comprises the effector binding loop (residues [30][31][32][33][34][35][36][37][38][39][40], β2 (residues 55 and 57), and several residues in α-helix 1. Pocket 2 involves the core β-strands 1 and 2, part of the effector loop, and switch 2.…”

Section: Resultsmentioning

confidence: 99%

“…4B). To test whether the ligands stabilize a particular Ras conformation, we used a principal component (PC)-based analysis developed previously (13,(34)(35)(36) to map conformers from K-Ras-SRJ23 trajectories onto a PC plane defined by crystal structures. Fig.…”

Section: Resultsmentioning

confidence: 99%

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

Hocker

Cho

Chen

et al. 2013

Self Cite

134

133

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several smallmolecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)-a bicyclic diterpenoid lactone isolated from Andrographis paniculata-and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP-GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP-GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.cancer | molecular dynamics | allosteric site | drug design

“…PCA, which is usually performed on Cartesian coordinates, may sometimes mask certain important long-range dynamical relations and complex features of biomolecular conformational dynamics. Comparing residue-residue contacts in various isoforms from difference contact maps built from simulation trajectories has aided in determining certain similar structural properties and some unique to a particular isoform (24). Another analysis that involves monitoring the time evolution of residue-residue contact formation and breaking has been proven useful in identifying certain characteristic events during conformational transitions (25).…”

mentioning

confidence: 99%

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Doshi

Holliday

Eisenmesser

et al. 2016

148

186

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.allostery | enzyme dynamics | residue-residue contacts | Cyclophilin A | molecular dynamics