A switch I mutant of Cdc42 exhibits less conformational freedom.

The success of target-based approaches to combat Ras-related signal transduction events leading to diseases such as cancer will depend upon understanding the correlations among structural biology, protein-protein interactions and protein function. Indeed, binding events and the activation of protein receptors may play a role in abnormal cell-signaling activities. Understanding such activities will require approaches that elucidate changes in both protein conformation and protein dynamics that lead to particular molecular events and behaviors. Cdc42 is a member of the Rho subfamily of Ras proteins and are closely related to small GTPases. These proteins are ubiquitously expressed, evolutionarily conserved, and among the first human oncogenic proteins to have been identified (1, 2). Mutations or abnormal expression patterns of Ras proteins have been found in up to 30% of cancer cell types (1, 2). Ras proteins act as molecular switches that couple extracellular signals to a variety of cellular responses. Ras protein activities are controlled via cycling between a biologically active GTP form and an inactive GDP-bound form (1, 3). The function of Cdc42, as well as other Ras proteins, is largely mediated through protein-protein interactions. Therefore, understanding the specificity governing these interactions, and associated conformational changes is vital for understanding molecular mechanisms underlying the onset of diseases such as cancer. Different signals can activate guanine nucleotide exchange factors (GEFs) that induce the small G-protein to switch from the inactive, GDP-bound state to the active, GTP-bound state (4, 5). In the active state, the GTPase binds downstream effector and the effector in turn can mediate diverse biological responses. These different signals are terminated when Cdc42 is deactivated, either through its own ability to hydrolyze GTP, or by means of a reaction catalyzed by GTPase-activating proteins (GAPs) (6). Alternatively, interactions with regulatory proteins that block GDP dissociation, such as GDP dissociation inhibitors (GDIs), stabilize the GDP-bound inactive state, and prevent effective GTP-GDP cycling (7). Mechanisms by which Cdc42, as well as most Ras proteins participate in cellular proliferation and/or transformation include mutations that alter the GTPase activity (1, 8–11) and mutations that alter the interactions with effectors or regulators (12). The best understood mutations have been those that result in cellular transformation by inhibiting GTPase activity (1, 8). In addition to this, Cdc42 mutations have also been shown to facilitate cell transformation by increasing the rate of cycling between active and inactive forms of the protein (13 199). Structural studies of the mutant Cdc42(F28L) provided evidence that local environmental changes affect the stability of the nucleotide-binding site leading to aberrant cell-signaling behavior (14). The binding of effector molecules to Cdc42 essentially involves two regions: Switch I (residues 28–40) and Switch II (residues 57–74), which can undergo rapid conformational changes. In fact, it has been reported that the Switch I region in H-Ras possesses two distinct conformational states, bound to effectors, and unbound (15, 16). Indeed, Switch I is the principal interacting surface for Cdc42 with downstream effectors (11, 17). For Cdc42 in solution, several resonances in the Switch regions cannot be observed due to increased flexibility in these regions (18). The binding of effectors to Cdc42 results in the loss of backbone mobility of the Switch I region, suggesting that the inherent dynamics may allow Cdc42 to bind to different effector proteins (19). It has been widely thought that the conformational freedom of the Switch I region may provide the basis for nucleotide-dependent signal propagation of Cdc42, and that the conformational flexibility of this region is central to the engagement of downstream effectors (17, 20, 21). This premise agrees with biological studies (22). Structural studies of Ras have implicated Thr35 in the Switch I region as being critical for the conformational changes as it is completely invariant in Ras related proteins. 31P-NMR studies with H-Ras(T35A) and Cdc42(T35A) suggested that this mutation causes a signaling-defective state (11, 16). The 31P-NMR spectra of both protein in their GTP-bound state revealed only small changes, suggesting that the nucleotide-binding site of the mutant was similar to wild type. However, binding peptide effectors that target the Switch I region produced significant shifts of 31P resonances in the wild type Ras peptide complexes but not in the T35A-effector complexes, suggesting a weaker interaction between effector peptide derivatives and the mutant protein (11, 16). To investigate the importance of the conformational flexibility in Switch I of Cdc42, we have determined the solution structure, examined the backbone dynamics, and characterized the biochemical activity of Cdc42(T35A) (2kb0.pdb). Our results suggest that the mutation of Thr35 leads to the loss of conformational freedom in Switch I relative to the rest of the protein. Our results furthermore provide molecular details on why the conformational flexibility of this region is inherently important for the recognition of the Ras-related effectors. Recent studies have implied that innovative strategies to target cancer treatment in the future should target specific mutant Ras proteins and directly block Ras protein interactions with oncogenic potential, for example using antibody fragments (23). Taken together, the structure and dynamics results for Cdc42(T35A) suggest that, in addition to strategies to block Ras-protein interactions, strategies to restrict the conformational flexibility of important binding regions of Ras leading to the inhibition of effector interactions that facilitate aberrant signaling activity may also have therapeutic potential.