NMR Structure and Mutagenesis of the N-Terminal Dbl Homology Domain of the Nucleotide Exchange Factor Trio

Liu, Xiaohong; Wang, Hong; Eberstadt, Matthias; Schnuchel, Arndt; Olejniczak, Edward T.; Meadows, Robert P.; Schkeryantz, Jeff M; Janowick, David A.; Harlan, John E.; Harris, Edith A. S.; Staunton, Donald E.; Fesik, Stephen W.

doi:10.1016/s0092-8674(00)81757-2

Cited by 168 publications

(175 citation statements)

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“…As has been seen for other DH domains (Liu et al, 1998), the exchange activity of the intersectin 1L DH domain alone is reduced relative to the exchange activity of the DHPH domains together (Figure 3C and Hussain et al, 2001). Phosphatidylinositol phosphates have been shown to bind GEF associated PH domains and in some instances this binding regulates the DH domain exchange activity (Han et al, 1998;Nimnual et al, 1998;Crompton et al, 2000;Das et al, 2000;Russo et al, 2001;Snyder et al, 2001).…”

Section: The In Vitro Gef Activity Of the Intersectin 1l Dh Domain Issupporting

confidence: 66%

“…Their conserved catalytic domain, the Dbl homology (DH) domain (Hart et al, 1991(Hart et al, , 1994 catalyzes the release of GDP from Rho GTPases and thus subsequent activation by GTP binding. DH domains are invariably found upstream and adjacent to pleckstrin homology (PH) domains, which are thought to influence their activity (Liu et al, 1998;Das et al, 2000) and membrane localization (Whitehead et al, 1999). The noncatalytic parts of the structurally complex GEFs link the exchange activity to cellular processes and inhibit the DH domain exchange activity (Zheng, 2001;Hoffman and Cerione, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

Zamanian

Kelly

2003

MBoC

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Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L. INTRODUCTIONRho family guanine nucleotide exchange factors (GEFs) are critical regulatory proteins of cellular pathways that require regulation of actin cytoskeleton rearrangements (for review see Zheng, 2001;Hoffman and Cerione, 2002). Their conserved catalytic domain, the Dbl homology (DH) domain (Hart et al., 1991(Hart et al., , 1994 catalyzes the release of GDP from Rho GTPases and thus subsequent activation by GTP binding. DH domains are invariably found upstream and adjacent to pleckstrin homology (PH) domains, which are thought to influence their activity (Liu et al., 1998;Das et al., 2000) and membrane localization (Whitehead et al., 1999). The noncatalytic parts of the structurally complex GEFs link the exchange activity to cellular processes and inhibit the DH domain exchange activity (Zheng, 2001;Hoffman and Cerione, 2002). Cellular inputs, such as protein (Hart et al., 1998;Scita et al., 1999;Innocenti et al., 2002) and phospholipid binding to (Han et al., 1998;Nimnual et al., 1998;Crompton et al., 2000;Das et al., 2000;Russo et al., 2001), and phosphorylation of (Crespo et al., 1997;Han et al., 1997;Schuebel et al., 1998;Aghazadeh et al., 2000) these regulatory regions derepress exchange activity. Integration of cellular signals by Rho GEFs can focus GTPase activity to allow temporal and spatially localized activation of actin cytoskeletal rearrangements.Intersectin 1L, a neuronal splice variant of the endocytic scaffolding protein intersectin 1, is a Rho family GEF that is composed of two N-terminal EH domains (EH1, EH2), a large region of putative coiled-coils, five SH3 domains (SH3A, B, C, D, and E; Roos and Kelly, 1998;Yamabhai et al., 1998;Okamoto et al., 1999;Sengar et al., 1999), followed by the DH and PH domains and a carboxyl-terminal ...

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Section: The In Vitro Gef Activity Of the Intersectin 1l Dh Domain Issupporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

Zamanian

Kelly

2003

MBoC

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“…The finding that the TrioN mutant N1406A/D1407A is inactive indicates that the DH domain is involved in the oxidase activation enhancing activity of TrioN. It has been shown that the N-terminal PH domain of Trio binds to acidic phospholipids and might serve as a membrane localizing signal (41). We cannot yet establish the possible involvement of the PH domain in the potentiating effect of Trio, but it is likely that a [TrioN-Rac1-GDP] complex will express a high affinity for the membrane because of the presence of two groups binding to acidic phospholipids, the prenylated polybasic C terminus of Rac1 and the PH domain of Trio.…”

Section: Discussionmentioning

confidence: 99%

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Sigal¹,

Gorzalczany²,

Sarfstein³

et al. 2003

Journal of Biological Chemistry

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The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b 559 and four cytosolic components as follows: p47 phox , p67 phox , p40 phox , and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b 559 and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67 phox but in the absence of amphiphile and p47 phox . We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67 phox -Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg 2؉ from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function. . 4). The identity of the cytosolic component(s) responsible for causing the conformational change in gp91 phox is controversial. The principal candidate is p67 phox , based on the identification of an "activation domain" in p67 phox , consisting of residues 199 -210 (5), and on direct evidence of binding of p67 phox to gp91 phox , an interaction enhanced by Rac1 (6). This hypothesis also implies that p47 phox and Rac serve either as "carriers" for p67 phox from the cytosol to the membrane or, following their own translocation, as membrane "anchors" for the correct positioning of p67 phox in the assembled complex. Oxidase assembly can be elicited in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac, exposed to an anionic amphiphile (7,8). The role of the amphiphile is to induce a conformational change in p47 phox , resulting in its binding to p22 phox and the passive translocation of p67 phox , by virtue of its affinity for p47 phox , to the vicinity of gp91 phox . The in vivo equivalent of amphiphile action is the phosphorylation of critical serines in p47 phox , a process representing the starting point of a "p47 phoxinitiated" pathway of oxidase activation.Under certain condi...

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“…[46]). Global folds can also be used as a structural stepping stone in the generation of high-resolution structures, since the assignment of additional NOEs from random fractionally deuterated samples or fully protonated molecules is facilitated by the use of a preliminary structure [47,48]. Further improvements in the quality of structures can also be obtained through the incorporation of additional restraints such as dipolar couplings ( [45]) or homology modeling (e. g. Ref.…”

Section: Structure Determination Of Selectively Methyl Protonated Promentioning

confidence: 99%

“…In cases where overlap in the aromatic spectrum becomes problematic, a synthetic strategy has been introduced to produce Phe that is 13 C labeled only at the epsilon position [52]. An illustration of the utlility of this approach is provided by the structure determination of a 21 kDa Dbl homology domain containing seven phenylalanine residues [47].…”

Section: Introducing 1 H 12 C Aromatic Residues Into Otherwise 13 C mentioning

confidence: 99%

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

Patzelt

Goto

Iwaï

et al. 2002

BioNMR in Drug Research

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The introduction of stable isotopes into proteins has significantly reduced the time requirements for structure elucidation of biomolecules. Moreover, structural studies of proteins with molecular weights exceeding the 10 kDa limit are usually not possible without uniform isotope labeling because of severe resonance overlap and inefficient coherence transfer along the rather small 3 J 1 H-1 H couplings. Nowadays, efficient expression of recombinant proteins is a prerequisite for many techniques used in structural biology, but the requirement for isotope labeling in particular often precludes NMR (nuclear magnetic resonance) structure determination of proteins isolated from natural sources. Specifically, proteins that have been uniformly labeled with 15 N and/or 13 C are commonly required for NMR spectroscopy, especially for backbone chemical shift assignment procedures, which are greatly facilitated by the use of a series of rather sensitive multi-dimensional triple-resonance NMR experiments (see Chapt. 4) [1], in a process that can also be automated with good success [2]. Moreover, the random replacement of nonexchangeable protons by deuterons reduces 1 H-1 H dipolar interactions and scalar couplings, thereby reducing peak line widths considerably and allowing structure elucidation of proteins exceeding 30 kDa [3,4]. Random fractionally deuterated protein samples also permit the use of longer mixing times in NOESY (nuclear Overhauser effect spectroscopy) experiments, since spin-diffusion pathways are largely eliminated. In addition, transverse-relaxation optimized spectroscopy (TROSY [5], see also Chapt. 10), which has been used for molecules larger than 100 kDa [6], benefits dramatically from deuteration. This stems from the fact that the TROSY component that is narrowed by the DD-CSA compensation is broadened by dipolar interactions with nearby protons.Besides uniform labeling approaches, stable isotopes can also be introduced at specific sites in proteins in order to simplify the assignment process and to isolate spectral information from the region of interest. For example, biosynthetically-directed fractional 13 C labeling offers the possibility of making stereospecific assignments of all isopropyl methyl groups of Val and Leu residues [7]. In another approach often used for solid-state NMR termed residue-specific labeling, isotope labels are introduced at single sites in a protein, as described in another chapter of this volume (Chapt. 11). A related scheme, called amino acid-type labeling, is accomplished by expression in an amino acid-based medium

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NMR Structure and Mutagenesis of the N-Terminal Dbl Homology Domain of the Nucleotide Exchange Factor Trio

Cited by 168 publications

References 50 publications

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

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