Here we investigate the structural and biochemical propertie
Here we investigate the structural and biochemical properties of the N-GTPase domain of p190RhoGAP-A. We determine the crystal structure and find that N-GTPase adopts an extended GTPase-like fold with six unique inserts that seem to preclude its ability to bind typical GAP, GEF, or Sulfamethazine mg molecules. We find that N-GTPase is bound constitutively to GTP/Mg2+ and lacks intrinsic catalytic activity. We also use mutational analysis to show that GTP and Mg2+ binding stabilizes the domain. Therefore, our study supports both that p190RhoGAP N-GTPase is a nucleotide binding, non-hydrolyzing, pseudoGTPase domain that cannot bind canonical GAP, GEF, or effector proteins, which may act as a protein-protein interaction module. Unique among known proteins, the p190RhoGAPs contain three pseudoGTPase domains.
Discussion The p190RhoGAP proteins are two of the most important regulators of Rho GTPase signaling. However, surprisingly, although these proteins were identified almost 30 years ago much remains to be learnt about their molecular level details. For example, recently we discovered that, within the putative unfolded “middle domain” of the p190RhoGAPs, there are two non-nucleotide binding pseudoGTPase domains (Stiegler and Boggon, 2017) (pG1 and pG2, Figure 1A). We therefore decided to probe the molecular level details of the N-terminal GTPase domain. In the present study, we show that this domain is a nucleotide binding, non-hydrolyzing, pseudoGTPase domain. Furthermore, our study strongly suggests that this domain cannot bind canonical GAP, GEF, or effector proteins in the usual manner. These results therefore firmly place the p190RhoGAP N-GTPase domain in the pseudoGTPase group. Pseudoenzymes corresponding to active enzymes that catalyze nucleotide reactions, such as pseudokinases and pseudoGTPases, can be classified into three groups: those that cannot bind nucleotide, those that can bind but have no or very weak catalytic activity, and those with retained catalytic activity (Murphy et al., 2014). These are classified as groups i, ii, and iii, respectively (Table 2). Our study shows that p190RhoGAP N-GTPase is a pseudoGTPase that retains nucleotide binding activity (Table 2). Furthermore, based on our crystal structure we can now properly identify the G motifs based on structure alignment (Figure 2), and we observe numerous changes to the G motifs. This study provides a comprehensive biophysical and biochemical analysis of the N-GTPase domain of p190RhoGAP proteins and clearly places the domain as an Rnd-like, group ii, pseudoGTPase (Table 2). Our study adds another member to the growing list of pseudoGTPases identified by sequence and/or structural analysis. Interestingly, the list contains both single-domain small pseudoGTPases (CENP-M, Rnd3, and Gem) (Basilico et al., 2014, Foster et al., 1996, Splingard et al., 2007), and now also includes pseudoGTPase domains in the multidomain p190RhoGAP proteins. This resembles the domain architectures of single-domain (H-Ras, RhoA) or multidomain (GGAPs) (Xia et al., 2003) GTPases, and implies a variety of different roles in signal transduction pathways. As the search for these proteins continues, it is clear that pseudoGTPases can have very low sequence identity with typical GTPases, so sequence analysis alone is hard to use to identify new members. Furthermore, even better-conserved pseudoGTPase domains such as N-GTPase can have structural features that are hard to identify in the absence of a structure. More robust secondary structure matching and structure prediction are clearly necessary to further reveal otherwise cryptic pseudoGTPase domains. As there are over 150 small GTPase superfamily proteins, we maintain that the list of pseudoGTPases will continue to grow. Finally, the current results establish a curious and exciting result, that p190RhoGAP proteins contain three validated pseudoGTPase domains. This is an extremely unusual domain composition. In the pseudokinase class, the JAKs are famous for containing both a catalytically active kinase domain and a pseudokinase domain; however, to our knowledge, no protein has been discovered that contains such a high number of pseudoenzyme domains. The reason for this architecture is unclear. We observe high conservation for all three pseudoGTPase domains through evolution, as far back as flies and sponges (Stiegler and Boggon, 2017). One intriguing possibility is that the N-GTPase could bind in cis to the GAP domain at the C terminus of p190RhoGAP to regulate its activity; however, our structural analysis predicts that this is very unlikely due to steric clashes (Figure 6). Therefore, although there seem to be important evolutionarily conserved functional reasons for maintenance of these domains, the functional roles are not yet identified. We postulate that N-GTPase acts as a protein-protein interaction scaffold or allosteric modulator. It is intriguing to further hypothesize that the unique surface on N-GTPase formed by the insert sequences provides the binding site for a potential protein partner. The presence, therefore, of three pseudoGTPase domains within the p190RhoGAP proteins provides an exciting precedent for the continued study of this exciting class of protein.