Solution Structure of the State 1 Conformer of GTP-bound H-Ras Protein and Distinct Dynamic Properties between the State 1 and State 2 Conformers

Abstract

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTPbound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and 31 P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an HRasT35S mutant in complex with guanosine 5-(␤, ␥-imido)-triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone 1 H, 15 N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras⅐GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.Small GTPases H-Ras, K-Ras, and N-Ras, collectively called Ras, are the products of the ras proto-oncogenes and function as molecular switches by cycling between the GTP-bound active and the GDP-bound inactive forms in intracellular signaling pathways controlling proliferation, differentiation, and apoptosis of cells. GTP hydrolysis on Ras is markedly stimulated by GTPase-activating proteins, whereas conversion from the GDP-bound form to the GTP-bound form is promoted by guanine nucleotide exchange factors (1, 2). Ras comprise the Ras family of small GTPases together with a number of its relatives including Rap1, Rap2, R-Ras, R-Ras2/TCL, M-Ras/RRas3, RalA, RalB, etc. (3). Structural studies of Ras showed that structural differences between the GDP-and GTP-bound forms universally exist in two flexible regions, called switch I (residues 32-38 in H-Ras) and switch II (residues 60 -75 in H-Ras) (1). GTP-sensitive orientation of the switch regions enables Ras to interact with their effectors such as Raf kinases and phosphoinositide 3-kinases (2). Recent 31 P NMR studies suggested that H-Ras in the nucleoside triphosphate form exists in equilibrium between two kinds of conformational states, state 1 and state 2, around the phosphate groups of GTP or its non-hydrolyzable analogues, GppNHp 3 and GTP␥S, bound to the protein (4 -6). This conformational heterogeneity has been commonly observed in a number of Ras homolo...

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

“…Gly12 is not spatially proximal to the membrane in either orientation (Fig. 4B); however, the G12D substitution was previously shown to increase the population of a nucleotide-free-like conformation of RAS-GTP called state 1, which increases the entropy (i.e., mobility) of the switch I/II and helix α3 region (17,18). The enhanced dynamics in the nucleotidebinding region (19) is most likely responsible for destabilizing the β-interface.…”

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

“…We used NMR spectroscopy to obtain structural information on the compound-binding interface on Ras·GTP. The NMR structure corresponding to the unique conformation of H-Ras·GppNHp was determined by using only its T35S mutant (13), because this mutation almost eliminated the slow conformational exchange process (14), which made NMR analysis of the wild-type protein impractical. Because of the low water solubility of Kobe0065 and Kobe2602, which made measurements of the NOEs impossible, we chose to use a water-soluble analog named Kobe2601 (Fig.…”

Crystal structures of the state 1 conformations of the GTP‐bound H‐Ras protein and its oncogenic G12V and Q61L mutants

“…Although crystal structures corresponding to state 2 were solved with wild-type (WT) H-Ras alone or in complex with the effectors [4,5], those corresponding to state 1 have only been solved with its mutants carrying T35S, G60A, and Y32F substitution [6][7][8] [6]. The solution structure of H-RasT35S-GppNHp also revealed polysterism in the switch regions, prominent in switch I [11].…”