Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Abstract

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the "insert region" (residues 122-135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.

Figure 1

The sequence of Cdc42 aligned with the sequence of K-Ras4B (top panel). The crystal structures of GNP-bound (PDB: 4JS0) and GDP-bound (PDB: 4DID) Cdc42 are shown (bottom panel). Although the sequence and most structural motifs are highly conserved, K-Ras4B is missing the insert region (residues 122–135), an α-helix rich in charged amino acids, which only appears in members of the Rho family. To see this figure in color, go online.

Figure 2

Snapshots depicting the final conformation in surface and cartoon representations, and superimposition of the final (magenta cartoon) and initial (pink cartoon) configurations of the wild type and two mutants of (A) the GTP-bound and (B) GDP-bound Cdc42. The insert region (light teal) loses its structural integrity in all the GDP-bound systems but not in the GTP-bound systems. To see this figure in color, go online.

Figure 3

The root mean-square fluctuations (RMSFs) for (A) the GTP-bound and (B) GDP-bound Cdc42 as a function of the residue number. The thick black bars denote the locations of the switch I and switch II regions, and the light-green background corresponds to the insert region in each figure. The backgrounds with light red and blue denote the β-sheet and α-helical secondary structures of the protein, respectively. Although the two switch regions and the insert region exhibit the largest fluctuations in all systems, the insert region fluctuates much more in the GDP-bound systems. The switch II region fluctuates more in the Cdc42^Q61L-GTP and Cdc42^G12V-GDP systems. To see this figure in color, go online.

Figure 4

Superimpositions of protein motion at the main normal mode with the lowest-frequency motion in stereo pair view for the wild-type and mutant GTP-bound (left column) and wild-type and mutant GDP-bound (right column) Cdc42. The images show the full range of motion for each residue. The motion clearly shows the loss of insert region integrity in the GDP-bound systems and the conformational flexibility of the switch I and switch II regions. Although the insert region has a visible range of motion in the GTP-bound wild-type and G12V mutant, it still maintains its helical structure.

Figure 5

The projection of the first three principal components, PC1, PC2, and PC3, for the (A) GTP-bound and (B) GDP-bound Cdc42. The PCA projection was subject to linkage clustering, and each cluster is shown in a different color. All the systems show three clusters, except the GTP-G12V mutant, which has two. Representatives of the clusters are shown in Fig. S2. To see this figure in color, go online.

Figure 6

Dynamic cross correlation map representing the covariance of residues between the switch I and insert region regions, and the switch II and insert region for the GTP-bound (left panels) and GDP-bound (right panels) Cdc42. The cross correlation values are depicted on a scale from blue to red. The figure shows big differences in the cross correlation between the GTP-bound and GDP-bound systems. In particular, the wild-type and G12V GTP-bound systems show stronger negative cross correlation between the insert region and the two switch regions (especially switch I). Weaker correlations are observed in the GTP-bound Q61L mutant. To see this figure in color, go online.

Figure 7

The potential of mean force (ΔG_PMF) along the reaction coordinates d₁ (defined by the distance from G60-Cα to GTP/GDP-P_β atom) and d₂ (defined by the distance from T35-Cα to GTP/GDP-P_β). As seen, the distances are very stable in the GTP-bound systems and the GDP-bound Q61L mutant. Both d₁ and d₂ fluctuate more in the GDP-bound G12V mutant, and they fluctuate to a lesser extent in the GDP-bound wild type. To see this figure in color, go online.

Figure 8

Comparison between Cdc42 and Rac1/Rac2 proteins. (A) Sequences of the insert region and its adjacent regions for Cdc42, Rac1, and Rac2. In the sequence, hydrophobic, polar/glycine, positively charged, and negatively charged residues are colored black, green, blue, and red, respectively. Underlining denotes the residues presenting the α-helical structure. The former is the helix-forming ¹¹⁷xDLRx¹²¹ motif, and the latter is the insert region. Snapshots representing the final structures of insert region for (B) GDP-bound (left panel) and GTP-bound (right panel) Cdc42 and (C) Rac1-GDP (left panel) and Rac2-GDP (right panel) are shown. Rac proteins preserved a highly stable insert region during a 1-μs simulation. In the structures, colors are the same as in the sequence, except for the hydrophobic resides with white. Dotted lines denote salt bridges. To see this figure in color, go online.