The structure of the Guanine Nucleotide Exchange Factor Rlf in complex with the small G-protein Ral identifies conformational intermediates of the exchange reaction and the basis for the selectivity

Abstract

CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf·Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.

Introduction

Small G-proteins are well recognised as molecular switches, which cycle between a GDP and a GTP bound state. Only the GTP bound state is able to interact with effector proteins to transmit the signal. Transition to the GDP bound state occurs via hydrolysis of the bound GTP by the intrinsic GTPase activity of the G-protein. This reaction is accelerated by GTPase activating proteins (GAPs). Guanine Nucleotide Exchange Factors (GEFs) catalyse the release of G-protein bound nucleotides and thereby allow rebinding of a new nucleotide. As the cellular concentration of GTP is higher than of GDP, the interaction of the GEF with the G-protein will predominantly result in GTP loading. The catalytic mechanism of the GEF reaction is based on a partial overlap of the binding interface for the nucleotide and for the GEF in the G-protein. In course of the exchange reaction, the GEF approaches the nucleotide bound G-protein such that a ternary complex is formed. In this complex the GEF is initially only loosely bound, but upon establishing its full binding interface with the G-protein the nucleotide is partially competed out of its binding site. Once loosely bound the nucleotide dissociates and a tight complex of GEF and G-protein is established. By reversing these steps a nucleotide can approach the complex of GEF and G-protein, such that a ternary complex is formed and compete out the GEF. Structural information is available on the stable binary complexes (Bos et al., 2007, Vetter and Wittinghofer, 2001) but little information is available of the intermediate conformational states during the transition between the stable binary complexes.

The Ras family of small G-proteins is constituted of 36 members and sub-grouped into to “the Ras-proteins” (H-Ras, K-Ras, N-Ras, M-Ras and R-Ras), “the Ral-proteins” (RalA and RalB), “the Rap-proteins” (Rap1A, Rap1B, Rap2A, Rap2B and Rap2C) and “the rest” (the remaining 24 members). “The Ras-proteins”, are established oncogenes and frequently found mutated in tumours (Karnoub and Weinberg, 2008). The Ral-GEFs Rgl1, Rgl2/Rlf, Rgl3 and RalGDS connect Ras signalling to “the Ral-proteins”. These GEFs contain a Ras Association (RA) domain at their C-terminus which binds specifically to the GTP bound form of Ras. This allows Ras·GTP to recruit the RalGEFs to membrane compartments where Ral is localised as well. “The Ral-proteins” are involved in the control of exocytosis (Moskalenko et al., 2002), endocytosis (Jullien-Flores et al., 2000), gene regulation (de Ruiter et al., 2000) and cellular transformation (Urano et al., 1996, White et al., 1996). They are in part responsible for the transforming phenotype of oncogenic Ras mutations and recently got attention in cancer therapy due to the development of Ral-targeting small molecules (Yan et al., 2014).

In addition to the RA domain at the C-terminus Ral-GEFs contain a REM domain and a CDC25-homology domain (CDC25-HD) at the N-terminus. REM domains co-occur with CDC25-HD (Quilliam et al., 2002). They mainly stabilise the fold of the CDC25-HD but the REM-domain of SOS, a Ras-GEF, is also involved in an allosteric regulation of exchange activity (Sondermann et al., 2004). The CDC25-HD mediates the guanine nucleotide exchange activity and is found in GEFs for G-proteins of the Ras-family. While GEFs for the Ras-, Rap-, and Ral-proteins are known, GEFs for “the rest” are unknown (Popovic et al., 2013b). Based on their specificity the GEFs are classified as Ras-, Rap- and Ral-GEFs. Ral-GEFs are assumed to be truly Ral-specific (Albright et al., 1993, Ferro et al., 2008, Wolthuis et al., 1997), while some Ras-/Rap-GEFs act on both Ras- and Rap-proteins and are thus crossing the subgroup border. The selectivity of Ras- and Rap-GEFs was systematically analysed and the amino acid residues in the G-proteins that determine selectivity were identified (Popovic et al., 2013b). The findings were rationalised based on the known crystal structures of CDC25-HD in complex with small G-proteins, the crystal structures of the Ras-GEF SOS1 in complex with H-Ras (Boriack-Sjodin et al., 1998) and the Rap-GEF Epac2 in complex with Rap1B (Rehmann et al., 2008). However, the selectivity of RalGEFs was not analysed and it is unclear how selectivity towards Ral-proteins is achieved. This aspect will be addressed here.