Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions

Abstract

Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.

Figure 1.

Sculpture of the canonical Ras-specific GTPase-activating protein (RasGAP) module showing the catalyitic portion central domain (GAPc) in red and the extra domain (GAPex) in green. (Artwork, by G. Haeusser, was created using foam rubber strips and acrylic on hard board [original size: 48″ × 35.4″].)

Figure 2.

Domain architecture of Ras-specific GTPase-activating proteins (RasGAPs). Representative RasGAPs are schematically shown with emphasis on their domain structure. Red boxes indicate GAP-(related) domains (GRDs). Sec14 represented in orange, src homology (SH, bronze), pleckstrin homology (PH, blue). Other domains like Ca²⁺-dependent phospholipid-binding/conserved region 2 (C2), isoleucine-glutamine repeat region (IQ), WW (conserved tryptophan), calponin homology (CH), juxtamembrane (JM), transmembrane (TM), and extracellular (EC) domains are also shown.

Figure 3.

Ras-specific GTPase-activating protein (RasGAP) structure and mechanism. (A) Ribbon representation of the Ras–RasGAP complex as derived from the structure of the catalytic domain of p120GAP/RASA1 (red and green) bound to Ras (yellow) and GDP-aluminum fluoride (AlF₃) (blue) (Scheffzek et al. 1997a), switch regions are shown in cyan, and arginine finger motif in violet. (B) Ribbon representation of different RasGAPs superimposed and modeled onto Ras shown in transparent yellow. View is rotated approximately 90° about a horizontal axis as compared to RasGAP in panel A representing a top view on the modeled complex. C2 domain fragment of SynGAP (blue) appears in proximity of the Ras-binding region in SynGAP. (C) Cartoon representation of the Ras–RasGAP complex including dual-specificity functionalities. Ras/Rap is shown in yellow and GAP in red. Upon binding of Ras/Rap to GAP, the GTPase activity is strongly enhanced by the complementation of the active site, delivering the catalytic arginine, 789 (p120GAP), or 711(PlexinC1). This is further stabilized by interaction with a secondary Arg/Lys in the GAP domain. Glutamine 61 of Ras or Glutamine 63 of Rap contributes to the catalysis reaction by positioning phosphate accepting water molecule for nucleophilic attack. Additional residues and motifs in GAP stabilize the switch I and switch II regions of Ras/Rap, supporting a conformation that is favorable for efficient GTP hydrolysis. (D) Close-up view of the Ras/Rap active site bound to GAP showing conformational concordance or variations in the switch regions on Ras-p120GAP and Rap-PlexinC1GAP. Components of the nucleotide-binding site are shown for Ras (beige) and Rap (pink). The catalytic arginines in PlexinC1GAP and p120GAP are included. (Structural visualizations in panels A, B, and D were done with The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.)

Figure 4.

Structural components of neurofibromin. (A) Ribbon representation of a putative Ras–GRD complex, modeled on the basis of the GAP-related domain (GRD) structure (Scheffzek et al. 1998a) and the Ras-p120GAP complex (Scheffzek et al. 1997a). Blue dots indicate the location of missense mutations found in neurofibromatosis type 1 (NF1) patients. The EVH1 domain of Spred1 shown to interact with extra domain (GAPex) is depicted in cyan. Below is the domain scheme of GRD of neurofibromin with amino-terminal and carboxy-terminal extra domains with nontruncating patient-derived mutations included (see text). (B) Ribbon diagram of the Sec14-PH module bound to lipid with approximately 90° rotation about a horizontal axis. Orange ribbon represents Sec14-like domain bound to lipid (phosphatidyl ethanolamine) (in violet). Pleckstrin homology (PH)-like domain is shown in blue. Helix represented in green is a part of Sec14 that forms lid helix. Red dots indicate the location of missense mutations found in NF1 patients. A tandem duplication and ΔK1750 are also shown. Below is the domain scheme of Sec14-PH of neurofibromin with nontruncating mutations identified in NF1 patients (see text). (C) Cartoon of the Sec14-PH module, illustrating the proposed structural changes associated with the exchange of glycerophospholipid ligands. Starting from the observed structure (1), lid helix (in green), and β-protrusion helix can move either stepwise (1 > 2 > 3) or in a concerted fashion (1 > 3) into an open conformation, which allows the exchange of lipid molecules between Sec14-PH and membrane (4). Binding of modulator to the lid helix or the β-protrusion appears to prevent the lipid exchange reaction (5). (Structural visualizations in panels A and B were done with The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.)

Figure 5.

Interaction partners of neurofibromin. Domain scheme of neurofibromin with reported interaction partners and structurally validated domains colored. Red dots at the bottom part of the scheme indicate nontruncating mutations found in patients and blue and yellow dots on the top part of the scheme represent potential phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC), respectively. Two ubiquitination sites are indicated as green boxes. Several potential caveolin-binding domains (CBDs) are also indicated. The GAP-related domain (GRD) part indicated in dark red is the minimal GAP domain required for the GAP activity and binds to Ras. Amino-terminal and carboxy-terminal extensions, indicated in light green are overlapping with the tubulin-binding region and the Sec14-PH domain, respectively, and interact with the Sprouty-related protein with an EVH1 domain 1 (Spred1). DDAH1, dimethylarginine dimethylaminohydrolase 1; LRPPRC, leucine-rich pentatricopeptide repeat-containing protein; APP, amyloid precursor protein; FAF2, Fas-associated factor 2; ETEA, expressed in T cells and eosinophils in atopic dermatitis; VCP, valosin-containing protein; LIMK2, LIM domain kinase 2; DPYSL2, dihydropyrimidinase-related protein 2; CRMP2/4, collapsin response mediator protein 2/4; SCF, Skp-Cullin-F box-containing complex; FAK, focal adhesion kinase; CASK, calcium/calmodulin-dependent serine protein kinase (see text).