# Roco Proteins: GTPases with a Baroque Structure and Mechanism
## 6 Roco Proteins: Conventional G-Proteins, GADs or Yet Another Type of G Proteins? Based on their working mechanism, G-proteins have been classified either as conventional G-proteins or G-proteins activated by dimerization (GADs) [108]. The catalytic cycle of G-proteins is dependent on both the rate of GTP hydrolysis itself and the rate of nucleotide exchange, i.e., the life-time of the nucleotide-free, GDP- and GTP-bound states. For conventional G-proteins, this cycle is highly regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). These regulators allow G-proteins to switch between an active and inactive state without unnecessary consumption of GTP [109; 110; 111; 97]. Conventional G-proteins typically have very high nucleotide affinities, in the nanomolar to picomolar range. This results in a very slow nucleotide dissociation [112; 97; 113]. Since in a cell, biological processes occur much faster, within seconds or minutes, than the intrinsic GTP dissociation, these proteins have co-evolved with GEFs. Binding of a GEF to its corresponding G-protein decreases the G-protein's affinity for the bound nucleotide making nucleotide dissociation possible. GEFs, thus, increase the nucleotide exchange rate (Figure 4a). Another level of regulation is added to the GTP hydrolysis cycle of conventional G-proteins by GAPs. Although conventional G-proteins are GTP hydrolysis enzymes, their actual GTP hydrolysis rate is intrinsically very low. In general, GAPs increase the rate of GTP hydrolysis by orienting a nucleophilic water molecule in an appropriate position for attack on the \(\gamma\)-phosphate of GTP and/or by stabilizing the negative charges of the transition state [109; 114]. In contrast to conventional G-proteins, GADs have low micromolar nucleotide affinities, leading to high nucleotide dissociation rates, making GEFs obsolete. Furthermore, when bound to GTP, the G-domains of GADs dimerize and, in this way, complement each other's active site. Each protomer, thus, provides residues for the active site of the adjacent protomer or has an important stabilizing role on its active site. These residues can stabilize the flexible active site or can be directly involved in catalysis. Either way, GADs have all the necessary components for GTP hydrolysis and are not strictly dependent on GAPs for GTP hydrolysis (Figure 4b) [108]. Figure 4: G-protein cycle of conventional G-proteins versus G-proteins activated by dimerization (GADs). (**a**) Conventional G-proteins have very high nucleotide affinities resulting in a low nucleotide dissociation rate. Binding of guanine nucleotide exchange factors (GEFs) decreases the affinity for the bound nucleotide allowing it to be released from the protein. Due to the higher cellular GTP concentration, GTP then binds to the protein and the protein switches to its active GTP-bound state, where it interacts with downstream effectors. GTPase activating proteins (GAPs) can then bind to the G-protein, stabilize and/or complement its catalytic machinery and in this way, increase its intrinsically low GTP hydrolysis rate. The G-protein then switches to its GDP-bound inactive state. (**b**) GADs have micromolar nucleotide affinities, leading to high nucleotide dissociation rates. Therefore, they do not require GEFs to cycle from their inactive GDP- to their active GTP-bound state. Following GTP binding, the G-domains of GADs dimerize. In this way, both subunits complement each other’s active site and are able to hydrolyse GTP. The GADs then cycle back to their inactive, GDP-bound state. In conclusion, GADs possess all the necessary components for GTP-binding and hydrolysis and cycle between an active and inactive state without the aid of GEFs and GAPs [108]. The exact GTPase mechanism of Roco proteins still forms the subject of debate due to the lack of structural information on the active GTP-bound conformation. In support of the conventional G-protein theory, some possible GEFs and GAPs have been reported for LRRK2 [115].