# Roco Proteins: GTPases with a Baroque Structure and Mechanism
## 6 Roco Proteins: Conventional G-Proteins, GADs or Yet Another Type of G Proteins?
Moreover, a wide variety of assays such as tandem affinity purifications, yeast two-hybrid assays, pull downs, co-immunoprecipitation, size-exclusion chromatography, single-particle transmission electron microscopy and immunogold labelling using cell extracts or purified LRRK2 confirmed a dimeric nature of LRRK2 under many circumstances [15; 56; 127; 128]. Gel filtration and blue native gels using cell lysates suggested that the majority of LRRK2 was dimeric, with a small fraction being monomeric or forming higher oligomers [82; 129]. Furthermore, it was shown that the GTPase activity of the full length LRRK2 (k\({}_{\text{cat}}\) = 0.5 min\({}^{-1}\); K\({}_{\text{M}}\) = 400 uM) or its RocCOR-kinase construct (k\({}_{\text{cat}}\) = 0.8 min\({}^{-1}\); K\({}_{\text{M}}\) = 343 uM) is 25 to 40 times higher compared to Roc alone (k\({}_{\text{cat}}\) = 0.02 min\({}^{-1}\); K\({}_{\text{M}}\) = 553 uM),suggesting that dimerization of LRRK2 indeed might increase its GTPase activity, although other factors can also contribute to the observed low activity of the isolated Roc domain [18; 105; 130]. Despite these pieces of evidence that purified Roco proteins behave mainly as dimers, cell fractionation experiments using endogenous or exogenous expressed LRRK2 revealed that the majority of LRRK2 in cells is cytosolic and monomeric, while only a small portion of dimeric LRRK2 is localized at the membrane. The membrane-associated dimeric LRRK2 shows a higher kinase activity, increased GTP-binding capacity and a decrease in phosphorylation [82; 129]. Moreover, the first study in living cells, using confocal and total internal reflection microscopy coupled to the number and brightness analysis, confirmed the monomeric nature of LRRK2 in the cytosol and the formation of higher oligomers in the plasma membrane [106; 131]. Recent work by our research groups further investigated the oligomeric state of Roco proteins during GTP hydrolysis in vitro. By combining small-angle X-ray scattering, size-exclusion chromatography coupled to multi-angle light scattering, sedimentation velocity analytical ultracentrifugation, native mass spectrometry and electron microscopy it was shown that CrRoco is mainly monomeric when bound to GTP, dimeric in its nucleotide-free state and that an intermediate state can be observed upon binding to GDP. Our very recently published structure of a LRR-RocCOR domain construct of CrRoco, in combination with hydrogen-deuterium exchange coupled to mass spectrometry analysis, revealed that these nucleotide-dependent changes in oligomerisation are relayed to conformational changes among the LRR, Roc, and COR domains via the Roc switch II [17]. The different oligomeric states are also observed during a round of GTP hydrolysis and form an integral part of the GTP hydrolysis cycle [19]. In parallel, a kinetic characterization of full length _Ct_Roco has demonstrated that neither nucleotide dissociation nor P\({}_{\text{i}}\) release are rate-limiting, but that GTP hydrolysis itself, or associated conformational changes after nucleotide binding but prior to product release, form the rate-limiting step in the GTP hydrolysis cycle [18].