Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras

Scheffzek, Klaus; Lautwein, Alfred; Kabsch, Wolfgang; Ahmadian, Mohammad Réza; Wittinghofer, Alfred

doi:10.1038/384591a0

Cited by 169 publications

(128 citation statements)

References 29 publications

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“…S1), the two GAP homologous regions C1 and C2 of plexin A3 form a GAP domain with an overall fold similar to canonical Ras GAPs such as p120 Ras GAP ( Fig. 1 B and C) (12,24,25). The two catalytically critical arginine residues in plexin A3, Arg-1407 and Arg-1724, superimpose precisely with those in p120 Ras GAP (Fig.…”

Section: Resultsmentioning

confidence: 91%

“…To address these questions we determined the crystal structure of the intracellular domain of plexin A3, one of the most widely expressed plexins in the nervous system (23). The structure shows that C1 and C2 of plexin fold in an intertwined manner to form a GAP domain that has an overall fold similar to other Ras GAPs (12). Structural comparisons suggest that the plexin GAP is in an autoinhibited state, and Rho GTPase binding alone does not appear to be able to induce its activation.…”

mentioning

confidence: 99%

“…The C1 and C2 regions each contain one of the two catalytic arginine residues (12), and the mechanism through which these two arginines are positioned precisely in close proximity for catalysis is not known (13). The domain between C1 and C2 in class A and B plexins binds Rho-family GTPases Rac1, Rnd1, or RhoD in their GTP-bound active form, and is called the Rho GTPase binding domain (RBD) (7,(14)(15)(16).…”

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confidence: 99%

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Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

He¹,

Yang

Terman

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Rac1 or Rnd1) to the RBD. Here we report the crystal structure of the plexin A3 intracellular region. The structure shows that the N-and C-terminal portions of the GAP homologous regions together form a GAP domain with an overall fold similar to other Ras GAPs. However, the plexin GAP domain adopts a closed conformation and cannot accommodate R-Ras/M-Ras in its substrate-binding site, providing a structural basis for the autoinhibited state of plexins. A comparison with the plexin B1 RBD/Rnd1 complex structure suggests that Rnd1 binding alone does not induce a conformational change in plexin, explaining the requirement of both semaphorin and a Rho GTPase for activation. The structure also identifies an N-terminal segment that is important for regulation. Both the N-terminal segment and the RBD make extensive interactions with the GAP domain, suggesting the presence of an allosteric network connecting these three domains that integrates semaphorin and Rho GTPase signals to activate the GAP. The importance of these interactions in plexin signaling is shown by both cell-based and in vivo axon guidance assays.autoinhibition ͉ axon guidance ͉ signaling ͉ structure

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Section: Resultsmentioning

confidence: 91%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

He¹,

Yang

Terman

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Modeling the RASA2 mutants on the structure of p120GAP predicts that R511 is a direct contact site for RAS (Fig. 4B) (33), and the NS-associated mutations are expected to damage RASA2 function (MAPP P = 2.12 × 10 − 5 , 9.42 × 10 − 5 , and 5.39 × 10 −6 , respectively).…”

Section: Significancementioning

confidence: 99%

Next-generation sequencing identifies rare variants associated with Noonan syndrome

Chen

Yin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

165

148

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Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed nextgeneration sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gainof-function alleles in Ras-like without CAAX 1 (RIT1) and mitogenactivated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that nextgeneration sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.human genetics | developmental diseases | whole exome sequencing | PTPN11 | RAS

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“…tures of the GTPase-activating domain of p120-GAP both alone and in complex with Ras have recently been published (4,5). It can be seen from these structures that the role of most of these conserved residues is likely to be structural.…”

mentioning

confidence: 99%

The Importance of Two Conserved Arginine Residues for Catalysis by the Ras GTPase-activating Protein, Neurofibromin

Sermon¹,

Lowe²,

Strom³

et al. 1998

Journal of Biological Chemistry

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Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 105 -fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg 1276 and Arg 1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras⅐GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate contributions of each of these residues to catalysis and binding affinity to Ras were measured. Both the R1276A and the R1391A mutant NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured at saturating concentrations. In contrast, there was only a minor effect of either mutation on NF1-334 affinity for wild-type Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely essential, because the mutant NF1-334 proteins increase the intrinsic Ras⅐GTPase by at least 100-fold. The roles of Arg 1276 and Arg 1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with Ras.

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Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras

Cited by 169 publications

References 29 publications

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

Next-generation sequencing identifies rare variants associated with Noonan syndrome

The Importance of Two Conserved Arginine Residues for Catalysis by the Ras GTPase-activating Protein, Neurofibromin

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