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Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.

Grant BJ, Gorfe AA, McCammon JA - PLoS Comput. Biol. (2009)

Bottom Line: The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function.In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras.Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, United States of America. bgrant@mccammon.ucsd.edu

ABSTRACT
Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25-40 and 57-75) intermediate between GTP and GDP states, or distinct loop3 (46-49), loop7 (105-110), and alpha5 C-terminus (159-166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of alpha2 (residues 66-74) with alpha3-loop7 (93-110), loop2 (26-37) with loop10 (145-151), and loop3 (46-49) with alpha5 (152-167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.

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Clustering of wild-type GTP with bound GDP (A–D) and wild-type GDP with bound GTP (E–H) aMD trajectories.Front and back views of representative structures obtained from hierarchical clustering (A, B, E and F). In each case the most populated cluster representative is shown in black (representative of 30.26% and 28.56% of their respective trajectory conformers in each simulation), with subsequent clusters in yellow (23.22% and 27.91%), green (21.3% and 21.02%), pink (19.29% and 16.34%) and red (5.94% and 6.17%). PC projection plots with cluster ellipsoid hulls i.e. the ellipsoid of minimum volume such that points from a given cluster lie inside ellipsoid boundaries (C and G). Trajectory timeline colored according conformational cluster (D and H).
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pcbi-1000325-g002: Clustering of wild-type GTP with bound GDP (A–D) and wild-type GDP with bound GTP (E–H) aMD trajectories.Front and back views of representative structures obtained from hierarchical clustering (A, B, E and F). In each case the most populated cluster representative is shown in black (representative of 30.26% and 28.56% of their respective trajectory conformers in each simulation), with subsequent clusters in yellow (23.22% and 27.91%), green (21.3% and 21.02%), pink (19.29% and 16.34%) and red (5.94% and 6.17%). PC projection plots with cluster ellipsoid hulls i.e. the ellipsoid of minimum volume such that points from a given cluster lie inside ellipsoid boundaries (C and G). Trajectory timeline colored according conformational cluster (D and H).

Mentions: Clustering of trajectory conformers was used to visualize the dominant conformations sampled by each simulation (Figure 2 and Table 1). The most populated cluster in the wild-type GDP-to-GTP transition (black in Figure 2E–H and Figure S4) corresponds to the lower of the three basins in Figure 1G and overlaps with the dominant conformation sampled during cMD simulations of the same system (Figure 1E). Its overall structure is intermediate between GDP and GTP states (RMSD of 1.3 Å from both GDP and GTP representatives, PDB codes 4q21 and 1qra respectively). Members of this cluster have an intermediate α2 orientation and a closed active site loop2-SI and loop4-SII that more closely resembles the GTP configuration. The second most populated cluster (yellow in Figure 2E–H) has more distinctive GTP like characteristics (RMSD of 1 Å from GTP and 1.6 Å from GDP representatives) including a closed loop2-SI and loop4-SII active site and a reoriented GTP-like α2 helix. Interestingly, the PC projection (Figure 2G) and RMSD values (minimum value 0.4 Å) indicate that members of cluster 2 closely resemble the crystallographic GTP-bound A59G structure (PDB code 1lf0). This structure has been suggested previously to be an intermediate and is characterized by a GTP-like SII conformation and a SI conformation that has undergone partial transition with the side-chain of Y32 adopting an orientation that is intermediate between that in wild-type GDP and GTP crystallographic structures [6],[7],[16] (see Figure S2). The third cluster (green in Figure 2E–H) is again equidistant from GDP and GTP states (RMSD of 1.3 to 1.4 Å) and, similar to cluster 1, is characterized by an intermediate α2 orientation. However, unlike cluster 1, loop2-SI and loop4-SII resemble the open GDP conformation. Clusters 4 and 5 are closer to the GDP conformation (with RMSDs of 1 and 0.3 Å) than to the GTP conformation (with RMSDs of 1.4 and 1.7 Å). However, cluster 5 has distinct conformations of loop3 and the C-terminal portion of α5.


Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.

Grant BJ, Gorfe AA, McCammon JA - PLoS Comput. Biol. (2009)

Clustering of wild-type GTP with bound GDP (A–D) and wild-type GDP with bound GTP (E–H) aMD trajectories.Front and back views of representative structures obtained from hierarchical clustering (A, B, E and F). In each case the most populated cluster representative is shown in black (representative of 30.26% and 28.56% of their respective trajectory conformers in each simulation), with subsequent clusters in yellow (23.22% and 27.91%), green (21.3% and 21.02%), pink (19.29% and 16.34%) and red (5.94% and 6.17%). PC projection plots with cluster ellipsoid hulls i.e. the ellipsoid of minimum volume such that points from a given cluster lie inside ellipsoid boundaries (C and G). Trajectory timeline colored according conformational cluster (D and H).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651530&req=5

pcbi-1000325-g002: Clustering of wild-type GTP with bound GDP (A–D) and wild-type GDP with bound GTP (E–H) aMD trajectories.Front and back views of representative structures obtained from hierarchical clustering (A, B, E and F). In each case the most populated cluster representative is shown in black (representative of 30.26% and 28.56% of their respective trajectory conformers in each simulation), with subsequent clusters in yellow (23.22% and 27.91%), green (21.3% and 21.02%), pink (19.29% and 16.34%) and red (5.94% and 6.17%). PC projection plots with cluster ellipsoid hulls i.e. the ellipsoid of minimum volume such that points from a given cluster lie inside ellipsoid boundaries (C and G). Trajectory timeline colored according conformational cluster (D and H).
Mentions: Clustering of trajectory conformers was used to visualize the dominant conformations sampled by each simulation (Figure 2 and Table 1). The most populated cluster in the wild-type GDP-to-GTP transition (black in Figure 2E–H and Figure S4) corresponds to the lower of the three basins in Figure 1G and overlaps with the dominant conformation sampled during cMD simulations of the same system (Figure 1E). Its overall structure is intermediate between GDP and GTP states (RMSD of 1.3 Å from both GDP and GTP representatives, PDB codes 4q21 and 1qra respectively). Members of this cluster have an intermediate α2 orientation and a closed active site loop2-SI and loop4-SII that more closely resembles the GTP configuration. The second most populated cluster (yellow in Figure 2E–H) has more distinctive GTP like characteristics (RMSD of 1 Å from GTP and 1.6 Å from GDP representatives) including a closed loop2-SI and loop4-SII active site and a reoriented GTP-like α2 helix. Interestingly, the PC projection (Figure 2G) and RMSD values (minimum value 0.4 Å) indicate that members of cluster 2 closely resemble the crystallographic GTP-bound A59G structure (PDB code 1lf0). This structure has been suggested previously to be an intermediate and is characterized by a GTP-like SII conformation and a SI conformation that has undergone partial transition with the side-chain of Y32 adopting an orientation that is intermediate between that in wild-type GDP and GTP crystallographic structures [6],[7],[16] (see Figure S2). The third cluster (green in Figure 2E–H) is again equidistant from GDP and GTP states (RMSD of 1.3 to 1.4 Å) and, similar to cluster 1, is characterized by an intermediate α2 orientation. However, unlike cluster 1, loop2-SI and loop4-SII resemble the open GDP conformation. Clusters 4 and 5 are closer to the GDP conformation (with RMSDs of 1 and 0.3 Å) than to the GTP conformation (with RMSDs of 1.4 and 1.7 Å). However, cluster 5 has distinct conformations of loop3 and the C-terminal portion of α5.

Bottom Line: The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function.In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras.Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, United States of America. bgrant@mccammon.ucsd.edu

ABSTRACT
Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25-40 and 57-75) intermediate between GTP and GDP states, or distinct loop3 (46-49), loop7 (105-110), and alpha5 C-terminus (159-166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of alpha2 (residues 66-74) with alpha3-loop7 (93-110), loop2 (26-37) with loop10 (145-151), and loop3 (46-49) with alpha5 (152-167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.

Show MeSH
Related in: MedlinePlus