Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

Jeffrey S Iwig,Jeffrey G Pelton,Rahul Das,Yvonne Vercoulen,Yan Che,Jeroen P Roose,David E Wemmer,Andre Limnander,Tiago Barros,John Kuriyan

doi:10.7554/elife.00813

Abstract

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI:http://dx.doi.org/10.7554/eLife.00813.001.

Highlights

An intriguing aspect of lymphocyte development is that the generation of a population of self-tolerant immune cells requires Ras activation by two distinct guanine nucleotide exchange factors, Ras guanine nucleotide releasing protein 1 (RasGRP1) and Son-of-Sevenless (SOS) (Figure 1A) (Dower et al, 2000; Ebinu et al, 2000; Kortum et al, 2011)
RasGRP1 is maintained in an autoinhibited, dimeric state, which is released by the coordinated binding of diacylglycerol and calcium second messenger molecules (Figure 12)
The N-terminal membrane-binding domains of SOS are coupled to this switch because they physically block access to the allosteric Ras-binding site

Summary

Introduction

An intriguing aspect of lymphocyte development is that the generation of a population of self-tolerant immune cells requires Ras activation by two distinct guanine nucleotide exchange factors, Ras guanine nucleotide releasing protein 1 (RasGRP1) and Son-of-Sevenless (SOS) (Figure 1A) (Dower et al, 2000; Ebinu et al, 2000; Kortum et al, 2011). The RasGRP and Ras guanine nucleotide releasing factor (RasGRF) families of exchange factors have tissue-specific expression patterns whereas SOS proteins are expressed ubiquitously (Stone, 2011). RasGRP proteins have been studied most extensively in T and B lymphocytes (Aiba et al, 2004; Brodie et al, 2004; Coughlin et al, 2005; Roose et al, 2005; Limnander et al, 2011) where they activate Ras in a manner that is non-redundant with SOS (Dower et al, 2000; Roose et al, 2007). RasGRP proteins play important roles in squamous cell carcinoma and melanoma (Luke et al, 2007; Oki-Idouchi and Lorenzo, 2007; Diez et al, 2009; Yang et al, 2011), T cell- and myeloid- leukemia

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Conclusion