# Roco Proteins: GTPases with a Baroque Structure and Mechanism
## 5 The Central RocCOR Domain Tandem of Roco Proteins: Structural Insights  
Roco proteins are characterized by a central RocCOR domain tandem, responsible for GTP binding and hydrolysis. Sequence alignments with other small GTPases and later crystal structures have shown that the overall architecture of the Roc domain is similar to the classical G-domain. This typical G-domain consists of a central \(\beta\)-sheet surrounded by \(\alpha\)-helices and connected by hydrophilic loops. Just as in other G-proteins, five G-domain sequence motifs (G1-5) can be discerned in the Roc domain, which are responsible for nucleotide binding: the phosphate binding loop or P-loop (G1), the switch I motif (G2), the switch II motif (G3), and the guanine specificity determining motifs (G4-G5) [97; 98; 99; 100; 101; 102; 103]. Unlike the first three sequence motifs, the guanine specificity determining motifs (G4-G5) show some variations compared to the classical G-domain [26]. In 2008, the crystal structure was reported of the isolated Roc domain of human LRRK2 in complex with GDP [104]. This structure showed a very unusual dimer, formed through extensive domain swapping (Figure 2). In this domain-swapped dimer, the N-terminal part (G1-3) of one subunit interacts with the C-terminal part (G4-5) of the other and vice versa, forming two hybrid nucleotide binding domains [104]. A later study of the Roc domain by [PERSON] et al., however, claimed this domain swapping to be an artefact of crystallization, since purification of an N- and C-terminally extended Roc domain yielded two peaks in size-exclusion chromatography corresponding to monomeric and dimeric LRRK2 Roc in solution [105]. Moreover, dimerization of the Roc domain was shown to enhance upon phosphorylation [106; 107]. A permanent dimerization of the Roc domains via domain swapping was also contradicted by subsequent crystal structures of the COR and RocCOR domain constructs of the Roco protein from the bacterium _Chlorobium tepidum_ (_Ct_Roco). Indeed, in 2008 and soon after the publication of the LRRK2 Roc crystal structure, a crystal structure of the RocCOR module of the _C. tepidum_ Roco protein in a nucleotide-free state was reported [33]. This structure also shows a dimeric arrangement while only one of the two Roc domains was resolved as an independently folded domain (Figure 3). In contrast, dimerization of the RocCOR in this structure was mainly mediated via interactions between the C-terminal part of the COR domains [33]. Moreover, structural alignment of the domain-swapped human LRRK2 Roc dimer with the bacterial _Ct_RocoCOR structure showed that domain swapping would result in severe clashes of the Roc domain with the COR domain, confirming that the Roc domains indeed cannot form a domain-swapped constitutive dimer in the context of the RocCOR [29; 105]. Further analysis of the COR and RocCOR structures of _Ct_Roco revealed that the COR domain actually consists of two subdomains connected by a flexible linker (Figure 3). The N-terminal half of the COR region (N-COR) is a mostly \(\alpha\)-helical domain with a short three-stranded antiparallel \(\beta\)-sheet. The C-terminal half of COR (C-COR) consists of an antiparallel \(\beta\)-sheet flanked by four \(\alpha\)-helices and a \(\beta\)-hairpin motif, the latter forming the main part of the COR dimerization interface [33]. As mentioned before, the crystal structure of _Ct_RocoCOR showed two COR domains forming the dimer interface, while only one Roc domain was resolved (Figure 3). The second Roc domain was thought to be present, but presumably, the electron density was very weak due to high mobility. The Roc domain has a typical canonical G-domain fold with an additional N-terminal helix (called \(\alpha 0\)), which in the context of the full-length protein would connect the Roc domain to the LRR domain. The switch I loop (G2) is flexible and is not visible in the structure. Moreover, sequence alignment of several Roco proteins demonstrated a large variability in length and amino acid composition of this motif.