Crystal structure and function of Rbj: A constitutively GTP-bound small G protein with an extra DnaJ domain

The Ras superfamily of GTPases are conserved in eukaryotes from yeast to humans, and play essential roles in the regulation of a variety of key cellular processes, including cell differentiation and proliferation, membrane trafficking, nuclear import and export, cytoskeletal remodeling and mitogenic signaling (Karnoub and Weinberg, 2008). Notably, numerous studies have shown that many members of the Ras-related small GTPases are involved in diverse aspects of tumorigenesis and tumor progression (Chen et al., 2014; Liu et al., 2017). These functions are a consequence of the GTP hydrolysis reaction catalyzed by a GTPase-domain (Gdomain), which can bind to and hydrolyze guanosine triphosphate (GTP) (“on” state) to guanosine diphosphate (GDP) (“off” state) . However, a small number of proteins naturally do not have the ability to hydrolyze GTP, yet play an important role in some diseases with unknown mechanisms (Chardin, 1999; Fiegen et al., 2002). To date, the Ras superfamily contains over 150 members, which can be divided into six subfamilies: Ras, Rho, Rab, Arf, Ran and RJL, based on sequence homology, structure and function (Goitre et al., 2014). The RJL family is an independent lineage of the Ras superfamily of GTPases, which can be classified into two distinct subfamilies: Rjl and Rbj (Nepomuceno-Silva et al., 2004). Besides the G-domain, members in this family contain an extra DnaJ domain. However, the function-structure relationship of these two domains and the function of the DnaJ domain are yet unclear. To understand the molecular basis behind their interactions of the latest identified small GTPase family members, the Xenopus laevis Rbj (xRbj) protein was expressed in Escherichia coli and its crystal structure has been determined to a resolution of 2.7 Å (Table S1). Two molecules were observed in one crystallographic asymmetric unit. While the 13 residues at the N-terminus are flexible, so each molecule contains 260 residues, spanning A14 to K273. The structure reveals that the G-domain and DnaJ domain are two independent domains, connected by a flexible hinge region (Fig. 1), thereby providing enough flexibility for binding effector proteins independently. Superimposing the G-domain of the two molecules in the asymmetric unit indicates that the two DnaJ domains are pointing to different directions, branching via the hinge region (Fig. S1), confirming the flexibility of the hinge region. The xRbj displays a typical GTPase fold consisting of a six-stranded β sheet (five parallel and one antiparallel) surrounded by five α helices, a typical Ras structure (Kityk et al., 2018) (Fig. S2A). One additional small helix is present between the α4 helix and β5 strand (Fig. 1B and 1C). The DnaJ domain consists of four α helices (Fig. 1B and 1C). In the GTPase active site, the Mg ion is tightly coordinated by the conserved residues S30 in P-loop, T48 in switch I, the γ-phosphate and β-phosphate atoms of the nucleotide and two well-ordered water molecules (Scheidig et al., 1999) (Fig. S2B). Unlike the strong GTPase activity of the Ras protein, xRbj remains in an “on” state, which has little, if any, GTPase activity (Fig. 2D). Previous studies indicated that Q61 in the switch II loop of Ras plays a key role in the intrinsic GTP hydrolysis reaction (Novelli et al., 2018) (Fig. 2A). Mutations of Ras at Q61 are known to slow the rate of GTP hydrolysis and transform healthy cells into malignant cells (Bos, 1989). The following residue E63 further stabilizes the position and orientation of Q61 by a hydrogen bond between the side chains (Fig. 2B). In contrast, while the counterpart residue H75 in xRbj possesses similar side chain length as Q61, the H75 side chain is oriented in the opposite direction of GTP, forming a hydrogen bond with the main chain of F77 (Fig. 2B). P76 is also more rigid than E62 in Ras, which further stabilizes the switch II loop. Consequently, H75 orients away from the γphosphate, which results in abrogation of GTPase activity (Fig. 2B). Therefore, we have defined xRbj G-domain as a GTP-bound protein. Site-directed mutagenesis experiments were performed to examine in more detail the contributions of the different residues to GTPase activity. H75Q (xRbj-H75Q) increased the GTPase activity of xRbj (Fig. 2D), which is consistent with previous reports (Chen et al., 2014). Triple mutations (xRbj-H75Q-P76E-F77E) on the switch II loop designed according to the structural analysis increased the hydrolysis activities to a greater extent, suggesting that these three residues are important for the lack of GTPase activity in


Construction of Rbj expression vectors.
The full-length X. laevis Rbj gene (xRbj, residues 1-273) was synthesized based on GenBank entry NC_Q7ZYF1 and codon optimized for expression in E.coli. The gene was cloned into the expression vector pET21a to form Rbj-pET21a, with a 6xHis tag at the C-terminus. For protein expression, the Rbj-pET21a plasmid was transformed into E. coli BL21 (DE3) competent cells. H75Q, A25G-E26G-H75Q, H75Q-P76E-F77E mutants were constructed based on Rbj-pET21a and expressed in the same way.

Expression and purification of Rbj proteins.
E. coli BL21 (DE3) cells carrying the Rbj-pET21a were grown in LB medium at 37℃ containing 50 mg/L ampicillin sodium. When the cell culture reached an optical density of A 600 =0.8, 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce protein expression. The cells were harvested 16 h post induction. The bacterial pellet was re-suspended by adding lysis buffer A (20 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM MgCl 2 ) and then lysed by passing through a microfluidizer twice.
The supernatant containing the soluble Rbj protein was passed through a 5 mL Ni-NTA column (GE Healthcare) pre-equilibrated with lysis buffer A for 1 h at 4℃.
The column was subsequently washed with buffer B (20 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 1 mM MgCl 2 ) for 20 column volumes. The Rbj protein was eluted with a solution of 300 mM imidazole, 20 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM MgCl 2 . The eluted Rbj protein was pooled and loaded onto a Superdex ® 75 16/60 column (GE Healthcare), which was equilibrated with buffer C (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MgCl 2 , 1 mM DTT). The resulting protein peak was concentrated to 15 mg/ml using a 30 kDa MWCO Amicon ® Ultra centrifugal concentrator. Aliquots were snap-frozen in liquid nitrogen and stored at -80℃ until they were used for crystallization. All the Rbj mutant proteins were prepared in the same way as the wildtype Rbj.

Crystallization and structural determination of Rbj.
For initial trial, Rbj was crystallized at 16℃ with vapor diffusion method in sitting Data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) BL19U.
Data were processed with HKL2000 (Otwinowski and Minor, 1997). The structure of Rbj was determined by molecular replacement with homologous structures (PDB 2A5J and 2YS8) as search models using the program Phaser (Read, 2001). The atomic model was rebuilt and completed with Coot (Emsley and Cowtan, 2004) and refined with phenix.refine (Adams et al., 2010). The stereochemical quality of the final model was assessed with PROCHECK (Laskowski R, 1993). Data collection and refinement statistics are summarized in Table S1. All structural figures were generated with PyMol (http://pymol.org). The atomic coordinate has been deposited in the Protein Data Bank. The PDB ID code is 6JMG.

Intrinsic GTPase activity essay
The intrinsic GTPase activity was monitored under the single-turnover condition adapting a strategy similar to Chandra et al (Chandra et al., 2014).

Circular dichroism (CD) spectroscopy.
The thermostability of wild type and mutant Rbj was tested by CD spectroscopy. The protein concentration was 0.2 mg/ml in 20 mM Tris (pH 8.0), 150 mM NaCl, 1mM MgCl 2 and 1mM DTT. Temperature-dependent CD experiments were analyzed at 205-260 nm from 20-80℃ with intervals of 1℃ to determine melting temperature (Tm). The data was analyzed using Prism 5 software. *Values in parentheses are given for the highest resolution shell.