Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Abstract

Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTPbound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5-(␤,␥-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the 31 P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.Small GTPases Ras (H-Ras, K-Ras, and N-Ras) are the products of the ras proto-oncogenes and presumed to be some of the most promising targets for anti-cancer drug development because of their high frequency of mutational activation in a variety of human cancers (1). Ras functions as a molecular switch by cycling between GTP-bound active and GDP-bound inactive forms in intracellular signaling pathways controlling cell growth and differentiation. Conversion between the GDPbound and the GTP-bound forms is controlled by guanine nucleotide exchange factors and GTPase-activating proteins (2, 3). Ras comprises the Ras family of small GTPases together with a number of its relatives, including Rap1, Rap2, R-Ras, R-Ras2/ TC1, M-Ras/R-Ras3, etc. (1). X-ray crystallographic and NMR analyses of H-Ras and Rap1A, alone or in complex with their effectors, revealed that the exchange of GTP for GDP results in allosteric conformational changes in two adjacent regions, termed switch I (residues 32-38) and switch II (residues 60 -75), and enables Ras to execute downstream signaling through direct interaction with its effectors, such as Raf kinases and phosphoinositide 3-kinases (2, 3). Recent 31 P NMR spectroscopic studies on Ras unveiled its novel structural feature, the conformational dynamics in the GTP-bound form (4). H-Ras and K-Ras in complex with Mg 2ϩ and a non-hydrolyzable GT...

H-Ras forms dimers on membrane surfaces via a protein–protein interface

“…The recombinant extension in the N terminus, either His-tags (49), large fluorescent proteins (20,50,51), or small oligopeptide tags for antibody staining (52), are generally considered to have little impact on biological functions (53)(54)(55). We find that a hexahistine tag on the N terminus of 6His-Ras(C181) slightly shifts the measured dimer K d (to 344 ± 28 molecules/μm 2 ) without changing the qualitative behavior of H-Ras dimerization (Fig.…”

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

“…GppNHp (16,6, and 6 mg/ml, respectively) were grown on seeds of a few pieces of finely pulverized crystals of H-RasT35S-GppNHp form 1 [6], which were transferred by using Disposable Crystal Probe manipulators (Hampton Research), at 20°C by the sitting drop vapor diffusion method using a reservoir solution containing 100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 200 mM ammonium sulfate, and 30% (w/v) PEG5000 MME. In addition, 1,5-diaminopentane dihydrochloride and b-nicotinamide adenine dinucleotide hydrate were added to the droplets of H-RasWT and Q61L, respectively.…”

“…Because association with the various effectors, such as cRaf-1, shifted the equilibrium toward state 2, state 1 and state 2 are regarded as ''inactive'' and ''active'' conformations, respectively [3]. 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].…”

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

“…When in state 1, Ras has reduced affinity for effectors and harbors open pockets (19,20), whereas in state 2, Ras is able to effectively bind effectors (21,22). In principle, small-molecule inhibitors that can selectively stabilize the state 1 conformation have the potential to inhibit Ras signaling by interfering with either effector or exchange factor binding.…”