Biomedical subjects

Alfred Wittinghofer

Publications and source records attributed to Alfred Wittinghofer.

At least 37 records · Page 2Linked to original sources

PubMed2005

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

Acylation↗

PubMed2005

The purification and crystallization of mDia1 in complex with RhoC.

An N-terminal construct of mouse mDia1 was recombinantly expressed in Escherichia coli, purified and crystallized in complex with truncated human RhoC using the hanging-drop vapour-diffusion method. Crystals were obtained using PEG 2K MME and MgSO4 as a precipitating agent and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 148.4, b = 85.2, c = 123.2 A. Complete native and SeMet-derivative data sets were collected at 100 K to 3.0 and 3.4 A resolution, respectively, using synchrotron radiation.

Animals↗

PubMed2005

Nucleotide binding and filament assembly of recombinant yeast septin complexes.

Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the gamma-phosphate.

Base Sequence↗

PubMed2004

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy.

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.

Amino Acid Sequence↗

PubMed2004

The structure of the TrmE GTP-binding protein and its implications for tRNA modification.

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.

Amino Acid Sequence↗

PubMed2004

Systematic peptide array-based delineation of the differential beta-catenin interaction with Tcf4, E-cadherin, and adenomatous polyposis coli.

Nuclear accumulation of the complex between beta-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on beta-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective beta-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the beta-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site.

Animals↗

PubMed2004

The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat.

The vasodilator-stimulated phosphoprotein (VASP) is a key regulator of actin dynamics. We have determined the 1.3-A resolution crystal structure of the 45-residue-long tetramerization domain (TD) from human VASP. This domain forms a right-handed alpha-helical coiled-coil structure with a similar degree of supercoiling as found in the widespread left-handed coiled coils with heptad repeats. The basis for the right-handed geometry of VASP TD is a 15-residue repeat in its amino acid sequence, which reveals a characteristic pattern of hydrophobic residues. Hydrophobic interactions and a network of salt bridges render VASP TD highly thermostable with a melting point of 120 degrees C.

Actins↗

PubMed2004

Synthesis of GTP-derived Ras ligands.

A practical and convenient method for the synthesis of acid- and base-sensitive GTP analogues carrying a further substituent at the terminal phosphate has been developed. Key to the successful synthesis of these potential ligands of the Ras protein is the use of Pd0-sensitive allyl protecting groups in a one-pot synthesis that avoids evaporation steps. Initial biochemical analysis of a representative compound revealed that such GTP analogues can bind to Ras and might open up the possibility of developing small molecules that can act as deactivators of oncogenic Ras.

Binding, Competitive↗

PubMed2004

Fourier transform infrared spectroscopy on the Rap.RapGAP reaction, GTPase activation without an arginine finger.

GTPase activating proteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004) Nature 429, 197-201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP(*) conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras.RasGAP reactions. However, the charge shift toward beta-phosphate observed with RasGAP was also observed for RapGAP. A GDP.P(i) intermediate accumulates in the GAP-catalyzed reaction, because the release of P(i) is eight times slower than the cleavage reaction, and significant GTP synthesis from GDP.P(i) was observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap.RapGAP catalytic machinery compensates for the absence of a cis-Gln by a trans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.

Arginine↗

PubMed2004

The GTPase-activating protein Rap1GAP uses a catalytic asparagine.

Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Galpha proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 A. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.

Adenosine Diphosphate↗

PubMed2004

Purification, crystallization and preliminary structural characterization of human Rap1GAP.

Human Rap1GAP, the GTPase-activating protein (GAP) for the small GTPase Rap1, was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Crystals were obtained using PEG 3350 as a precipitating agent and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 170.7, b = 224.5, c = 48.7 A. A complete data set was collected to 2.9 A resolution at 100 K using synchrotron radiation. The structure may reveal features of the unique reaction mechanism of Rap1GAP.

Cloning, Molecular↗

PubMed2004

The core FH2 domain of diaphanous-related formins is an elongated actin binding protein that inhibits polymerization.

Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerization of unbranched actin filaments. They contain three formin homology domains. Evidence as to the effect of formins on actin polymerization were obtained using FH2/FH1 constructs of various length from different Drfs. Here we define the core FH2 domain as a proteolytically stable domain of approximately 338 residues. The monomeric FH2 domains from mDia1 and mDia3 inhibit polymerization of actin and can bind in a 1:1 complex with F-actin at micromolar concentrations. The X-ray structure analysis of the domain shows an elongated, crescent-shaped molecule consisting of three helical subdomains. The most highly conserved regions of the domain span a distance of 75 A and are both required for barbed-end inhibition. A construct containing an additional 72 residue linker has dramatically different properties: It oligomerizes and induces actin polymerization at subnanomolar concentration.

Actins↗

PubMed2003

Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac.

Epac is a cAMP-dependent exchange factor for the small GTP-binding protein Rap. The activity of Epac is inhibited by a direct interaction between the C-terminal helical part of the cAMP-binding domain, called the lid, and the catalytic region, which is released after binding of cAMP. Herein, we show that the activation properties are very sensitive to modifications of the cyclic nucleotide. Some analogues are inhibitory and others are stimulatory; some are characterized by a much higher activation potential than normal cAMP. Mutational analysis of Epac allows insights into a network of interactions between the cyclic nucleotides and Epac. Mutations in the lid region are able to amplify or to attenuate selectively the activation potency of cAMP analogues. The properties of cAMP analogues previously used for the activation of the cAMP responsive protein kinase A and of 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate, an analogue highly selective for activation of Epac were investigated in detail.

Amino Acid Motifs↗

PubMed2003

Amino acids of the bacterial toxin SopE involved in G nucleotide exchange on Cdc42.

RhoGTPases are central switches in all eukaryotic cells. There are at least two known families of guanine nucleotide exchange factors that can activate RhoGTPases: the Dbl-like eukaryotic G nucleotide exchange factors and the SopE-like toxins of pathogenic bacteria, which are injected into host cells to manipulate signaling. Both families have strikingly different sequences, structures, and catalytic core elements. This suggests that they have emerged by convergent evolution. Nevertheless, both families of G nucleotide exchange factors also share some similarities: (a) both rearrange the G nucleotide binding site of RhoGTPases into virtually identical conformations, and (b) two SopE residues (Gln-109SopE and Asp-124SopE) engage Cdc42 in a similar way as equivalent residues of Dbl-like G nucleotide exchange factors (i.e. Asn-810Dbs and Glu-639Dbs). The functional importance of these observations has remained unclear. Here, we have analyzed the effect of amino acid substitutions at selected SopE residues implicated in catalysis (Asp-124SopE, Gln-109SopE, Asp-103SopE, Lys-198SopE, and Gly-168SopE) on in vitro catalysis of G nucleotide release from Cdc42 and on in vivo activity. Substitutions at Asp-124SopE, Gln-109SopE, and Gly-168SopE severely reduced the SopE activity. Slight defects were observed with Asp-103SopE variants, whereas Lys-198SopE was not found to be required in vitro or in vivo. Our results demonstrate that G nucleotide exchange by SopE involves both catalytic elements unique to the SopE family (i.e. 166GAGA169 loop, Asp-103SopE) and amino acid contacts resembling those of key residues of Dbl-like guanine nucleotide exchange factors. Therefore, besides all of the differences, the catalytic mechanisms of the SopE and the Dbl families share some key functional aspects.

Actins↗

PubMed2003

Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac.

Epac1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 that is directly activated by cAMP. This protein consists of a regulatory region with a cAMP-binding domain and a catalytic region that mediates the GEF activity. Epac is inhibited by an intramolecular interaction between the cAMP-binding domain and the catalytic region in the absence of cAMP. cAMP binding is proposed to induce a conformational change, which allows a LID, an alpha-helix at the C-terminal end of the cAMP-binding site, to cover the cAMP-binding site (Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2003) Nat. Struct. Biol. 10, 26-32). Here we show that mutations of conserved residues in the LID region affect cAMP binding only marginally but have a drastic effect on cAMP-induced GEF activity. Surprisingly, some of the mutants have an increased maximal GEF activity compared with wild type. Furthermore, mutation of the conserved VLVLE sequence at the C-terminal end of the LID into five alanine residues makes Epac constitutively active. From these results we conclude that the LID region plays a pivotal role in the communication between the regulatory and catalytic part of Epac.

Allosteric Regulation↗

PubMed2003

Structural view of a fungal toxin acting on a 14-3-3 regulatory complex.

The fungal phytotoxin fusicoccin stabilizes the interaction between the C-terminus of the plant plasma membrane H(+)-ATPase and 14-3-3 proteins, thus leading to permanent activation of the proton pump. This results in an irreversible opening of the stomatal pore, followed by wilting of plants. Here, we report the crystal structure of the ternary complex between a plant 14-3-3 protein, fusicoccin and a phosphopeptide derived from the C-terminus of the H(+)-ATPase. Comparison with the corresponding binary 14-3-3 complexes indicates no major conformational change induced by fusicoccin. The compound rather fills a cavity in the protein-phosphopeptide interaction surface. Isothermal titration calorimetry indicates that the toxin alone binds only weakly to 14-3-3 and that peptide and toxin mutually increase each others' binding affinity approximately 90-fold. These results are important for herbicide development but might have general implications for drug development, since rather than inhibiting protein-protein interactions, which is difficult to accomplish, it might be easier to reverse the strategy and stabilize protein-protein complexes. As the fusicoccin interaction shows, only low-affinity interactions would be required for this strategy.

14-3-3 Proteins↗

PubMed2003

Characterisation of PDZ-GEFs, a family of guanine nucleotide exchange factors specific for Rap1 and Rap2.

PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.

3T3 Cells↗

PubMed2003

Structure and regulation of the cAMP-binding domains of Epac2.

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.

Amino Acid Sequence↗