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Biomedical subjects

A Wittinghofer

Publications and source records attributed to A Wittinghofer.

At least 37 records · Page 2Linked to original sources
PubMed2000

Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism.

The interferon-gamma-induced guanylate-binding protein 1 (GBP1) belongs to a special class of large GTP- binding proteins of 60-100 kDa with unique characteristics. Here we present the structure of human GBP1 in complex with the non-hydrolysable GTP analogue GppNHp. Basic features of guanine nucleotide binding, such as the P-loop orientation and the Mg(2+) co-ordination, are analogous to those of Ras-related and heterotrimeric GTP-binding proteins. However, the glycosidic bond and thus the orientation of the guanine base and its interaction with the protein are very different. Furthermore, two unique regions around the base and the phosphate-binding areas, the guanine and the phosphate caps, respectively, give the nucleotide-binding site a unique appearance not found in the canonical GTP-binding proteins. The phosphate cap, which constitutes the region analogous to switch I, completely shields the phosphate-binding site from solvent such that a potential GTPase-activating protein cannot approach. This has consequences for the GTPase mechanism of hGBP1 and possibly of other large GTP-binding proteins.

Crystallography, X-Ray↗
PubMed2000

Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs.

Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.

Adenosine Diphosphate↗
PubMed2000

Priming of PC12 cells for semiquantitative microinjection studies involving Ras.

Nerve growth factor and activated Ras can induce differentiation of rat pheochromocytoma cells (PC12 cells) [Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73, 2424-2428] from a chromaffin cell-like morphology into one that resembles sympathetic neurones. We developed a special treatment of PC12 cells which apparently synchronizes these cells such that they are more useful for semi-quantitative microinjection studies for signal transduction pathways. This treatment leads to a faster and more reproducible differentiation which faithfully reproduces the involvement of Ras in the process and allows a comparison of the biological activity of different Ras mutants. It shows that G12V and Q61L oncogenic mutants are not equally potent in inducing differentiation. Partial loss-of-function mutations T35S, E37G and Y40C are inactive and even a triple combination of these does not restore full biological activity.

Amino Acid Substitution↗
PubMed2000

Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins.

Interferon-gamma is an immunomodulatory substance that induces the expression of many genes to orchestrate a cellular response and establish the antiviral state of the cell. Among the most abundant antiviral proteins induced by interferon-gamma are guanylate-binding proteins such as GBP1 and GBP2. These are large GTP-binding proteins of relative molecular mass 67,000 with a high-turnover GTPase activity and an antiviral effect. Here we have determined the crystal structure of full-length human GBP1 to 1.8 A resolution. The amino-terminal 278 residues constitute a modified G domain with a number of insertions compared to the canonical Ras structure, and the carboxy-terminal part is an extended helical domain with unique features. From the structure and biochemical experiments reported here, GBP1 appears to belong to the group of large GTP-binding proteins that includes Mx and dynamin, the common property of which is the ability to undergo oligomerization with a high concentration-dependent GTPase activity.

Amino Acid Sequence↗
PubMed2000

Bioorganic synthesis of lipid-modified proteins for the study of signal transduction.

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.

Animals↗
PubMed1999

PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2.

The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.

Amino Acid Sequence↗
PubMed1999

Selection of phage-displayed fab antibodies on the active conformation of ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras.

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effectors like c-Raf kinase, leading to cell growth. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecules that prevent downstream signaling may be of interest. Here, we show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al. (1999) J. Biol. Chem. 274, 18218-18230], a Fab antibody (Fab H2) was identified that exclusively binds to active Ras, and not to inactive Ras. Using surface plasmon resonance (SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to the homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilized Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC(50) value of 135 nM. This value compares well to the K(D) of active Ras-binding to immobilized Raf-RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, which does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping studies using SPR analysis demonstrate that the binding sites for the antibodies are non-identical. This antibody could be used for dissecting functional differences between Ras effectors. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies, and thus could be used for gene therapy of cancer with intracellular antibodies.

Antibodies↗
PubMed1999

Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.

RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopE.CDC42 complex are in the same order of magnitude as those described for complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopE.CDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 10(5)-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs.

Bacterial Proteins↗
PubMed1999

Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.

It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.

Cell Cycle Proteins↗
PubMed1999

Model of the ran-RCC1 interaction using biochemical and docking experiments.

RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.

Alanine↗
PubMed1999

Effector recognition by the small GTP-binding proteins Ras and Ral.

The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

ATP-Binding Cassette Transporters↗
PubMed1999

Guanosine triphosphatase stimulation of oncogenic Ras mutants.

Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.

Benzophenones↗
PubMed1999

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.

Amino Acid Sequence↗
PubMed1999

Structural and biochemical analysis of Ras-effector signaling via RalGDS.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.

Crystallography, X-Ray↗
PubMed1999

Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

Amino Acid Sequence↗
PubMed1999

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition.

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.

Amino Acid Sequence↗
PubMed1999

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport.

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.

Amino Acid Sequence↗