Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase

Abstract

Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein-protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295-304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many (1)H(N), (13)C, and (15)N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N-H correlations in the (1)H-(15)N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77-83 and residues 127-131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

“…Ab initio NMR assignment of small GTPases are typically challenging projects (e.g. Cao et al (102). Because the chemical shift perturbations of most resonances were small from the published NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4 (46), assignments could be transferred with high confidence for the dispersed signals.…”

K-Ras G-domain binding with signaling lipid phosphoinositides: PIP2 association, orientation, function

“…NMR assignment of small GTPases are typically challenging projects [e.g. Cao et al, 2013]. Thus, assignments were transferred, with a reasonable level of confidence for dispersed peaks, from the published NMR assignment for human K-Ras (residues 1-166) in the GDPbound form at a physiological pH of 7.4 [Vo et al, 2013].…”

“…1A). Lobe 1, the catalytic subdomain, encompassing residues 1-86, contains the functionally critical switch regions whose conformation and dynamics is nucleotide dependent (switch1 including residues 25-40, and switch 2 including residues [57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75], as well as the phosphate binding region (Ploop, residues [10][11][12][13][14][15][16][17]. The second lobe, comprised of residues 87-166, is a regulatory domain and contains allosteric regions such as helix 3 and loop 7.…”