Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Renault, Louis; Guibert, Bernard; Cherfils, Jacqueline

doi:10.1038/nature02197

Cited by 300 publications

(374 citation statements)

References 43 publications

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“…Communication between the membrane and the GEF domain involves little direct contact, if any, and can thus be described in the framework of allostery. It is well established that Sec7 domains have little conformational flexibility, 26,27,[29][30][31][32][33] which makes it unlikely that the GEF activity is regulated by conformational changes at the level of the Sec7 domain. In the following sections, we discuss our current knowledge on the regulatory roles of non-catalytic domains of eukaryotic and bacterial ArfGEFs at the membrane interface.…”

Section: The Allosteric Contribution Of Membranes To the Activation Omentioning

confidence: 99%

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Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

2016

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Arf GTPases assemble protein complexes on membranes to carry out major functions in cellular traffic. An essential step is their activation by guanine nucleotide exchange factors (GEFs), whose Sec7 domain stimulates GDP/GTP exchange. ArfGEFs form 2 major families: ArfGEFs with DCB, HUS and HDS domains (GBF1 and BIG1/BIG2 in humans), which act at the Golgi; and ArfGEFs with a Cterminal PH domain (cytohesin, EFA6 and BRAG), which function at the plasma membrane and endosomes. In addition, pathogenic bacteria encode an ArfGEF with a unique membrane-binding domain. Here we review the allosteric regulation of Arf GTPases and their GEFs at the membrane interface. Membranes contribute several regulatory layers: at the GTPase level, where activation by GTP is coupled to membrane recruitment by a built-in structural device; at the Sec7 domain, which manipulates this device to ensure that Arf-GTP is attached to membranes; and at the level of noncatalytic ArfGEF domains, which form direct or GTPase-mediated interactions with membranes that enable a spectacular diversity of regulatory regimes. Notably, we show here that membranes increase the efficiency of a large ArfGEF (human BIG1) by 32-fold by interacting directly with its Nterminal DCB and HUS domains. The diversity of allosteric regulatory regimes suggests that ArfGEFs can function in cascades and circuits to modulate the shape, amplitude and duration of Arf signals in cells. Because Arf-like GTPases feature autoinhibitory elements similar to those of Arf GTPases, we propose that their activation also requires allosteric interactions of these elements with membranes or other proteins.

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Section: The Allosteric Contribution Of Membranes To the Activation Omentioning

confidence: 99%

“…2C). Crystallographic analysis of Arf/ArfGEF intermediates captured by the drug BFA, 26,27 by a mutation of catalytic glutamate that occupies the Mg 2C binding site 26,28,29 and by removal of GDP 14 revealed that ArfGEFs activate membrane-associated Arf-GDP in 2 discrete steps (Fig. 2C).…”

Section: The Allosteric Contribution Of Membranes To the Activation Omentioning

confidence: 99%

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

2016

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“…One such mutation in yeast Gea1p, M699L, was able to confer resistance to BFA both in vivo and in vitro (Peyroche et al, 1999). The crystal structures of the Sec7 domain-Arf1-GDP-BFA complex have shown that M699 in Gea1 (corresponding to M832L in GBF1) makes a direct van der Waals contact with the BFA molecule in the complex (Mossessova et al, 2003;Renault et al, 2003). We introduced the corresponding mutation, M832L, into YFP-GBF1.…”

Section: Mechanism Of Bfa-induced Stabilization Of Gbf1 On Membranesmentioning

confidence: 99%

Dynamics of GBF1, a Brefeldin A-Sensitive Arf1 Exchange Factor at the Golgi

Niu

Pfeifer

Lippincott‐Schwartz

et al. 2005

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Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport. The mechanism of GBF1 binding to membranes is unknown. As a first step to understanding the mechanism of membrane association, we constructed a yellow fluorescent protein-tagged version of GBF1 and performed fluorescence recovery after photobleaching analysis to determine its residence time on Golgi membranes. We find that GBF1 molecules are not stably associated with the Golgi but rather cycle rapidly on and off membranes. The drug brefeldin A (BFA), an uncompetitive inhibitor of the exchange reaction that binds to an Arf-GDP-Arf GEF complex, stabilizes GBF1 on Golgi membranes. Using an in vivo assay to monitor Arf1-GTP levels, we show that GBF1 exchange activity on Arf1 is inhibited by BFA in mammalian cells. These results suggest that an Arf1-GBF1-BFA complex is formed and has a longer residence time on Golgi membranes than GBF1 or Arf1 alone.

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“…Precedent for this approach emerges from the observation that the natural product Brefeldin A (BFA) binds to a related small GTPase, Arf, and its exchange factor, ARNO. 9 In this complex, GDP is also present, resulting in a quaternary complex where BFA is located in a hydrophobic pocket at the Arf-ARNO interface, onethird of which is contributed by ARNO and two-thirds by Arf. BFA acts as an uncompetitive inhibitor that stabilises an abortive Arf-GDP-ARNO complex resulting in inhibition of the secretory pathway in eukaryotic cells.…”

Section: Introductionmentioning

confidence: 99%

Small Molecule Binding Sites on the Ras:SOS Complex Can Be Exploited for Inhibition of Ras Activation

et al. 2015

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Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPaseactivating protein-catalysed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilising or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilisation hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf, and is effective at inhibiting the exchange of labelled GDP in both mutant (G12C and G12V) and wild type Ras.

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Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Cited by 300 publications

References 43 publications

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

Dynamics of GBF1, a Brefeldin A-Sensitive Arf1 Exchange Factor at the Golgi

Small Molecule Binding Sites on the Ras:SOS Complex Can Be Exploited for Inhibition of Ras Activation

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