Search PubMed⌕ Search

Biomedical subjects

A Wittinghofer

Publications and source records attributed to A Wittinghofer.

At least 127 records · Page 7Linked to original sources
PubMed1993

Three-dimensional structure and properties of wild-type and mutant H-ras-encoded p21.

Ras (or p21) is the product of the ras proto-oncogene and is believed to be involved in growth-promoting signal transduction. The structure of the guanine nucleotide-binding domain of H-Ras (or p21H-ras) in the triphosphate conformation was determined at very high resolution (1.4 A). All the binding interactions between protein and Gpp[NH]p and Mg2+ can be resolved in great detail. The region around amino acids 61-65 is flexible and exists in two conformations, one of which seems to be important for catalysis. The properties and structures of several oncogenic and non-oncogenic mutant forms of Ras have also been determined. Since the structure of the GDP-bound form is also known, the nature of the conformational change from the GTP-bound to the GDP-bound form can be inferred from the 3-D structure. A mechanism for the intrinsic GTP hydrolysis has been proposed. Its implications for the GAP-stimulated GTPase reaction is discussed in the light of recent kinetic and mutational experiments.

Animals↗
PubMed1993

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons.

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal-transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF-induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.

Animals↗
PubMed1992

Binding of the H-ras p21 GTPase activating protein by the activated epidermal growth factor receptor leads to inhibition of the p21 GTPase activity in vitro.

There is strong, albeit indirect, evidence for a mitogenic signal transduction pathway comprising growth factors, growth factor receptors, the GTPase activating protein (p120-GAP), and p21ras. To demonstrate a direct physical association between these proteins in the absence of other cell constituents, their interaction was studied in vitro. Our results obtained with homogeneous protein preparations show that the activated epidermal growth factor (EGF) receptor phosphorylates p120-GAP at one site. Phosphorylated p120-GAP remains firmly bound to the receptor at physiological salt concentration; this leads to product inhibition of the receptor kinase activity as shown by diminished autophosphorylation activity and lack of turnover in p120-GAP phosphorylation. Phosphorylated p120-GAP is as active in stimulating the p21ras.GTPase as unphosphorylated GAP. p120-GAP, however, when bound to the EGF receptor is by a factor of 2 less active in stimulating the p21ras.GTPase than free p120-GAP. This effect might contribute to regulate the steady-state level of p21-GTP.

Enzyme Activation↗
PubMed1992

Simulation of the solution structure of the H-ras p21-GTP complex.

An unconstrained simulation of the GTP-bound form of the H-ras protein p21 is performed in an aqueous environment with charge-neutralizing counterions. The simulation is compared to the 1.35-A structure of Pai et al. [(1990) EMBO J. 9, 2351] and a proposed alternate structure, in which the loop at residues 60-65 is modeled into a form which may activate a water molecule for the GTP hydrolysis. The simulation suggests that some protein intermolecular H-bond contacts which are present in the crystal structure are lost in the solvation process and this loss may lead to localized refolding of the molecule. For instance, we find that the gamma-phosphate of the GTP has somewhat weaker contact with the protein in the simulation structure. The antiparallel beta-sheet (residues 38-57) partially melts. The 60-65 loop, which is hypervariable in the X-ray study, is initially relatively distant from the gamma-phosphate region. However, this loop moves so as to sample the space around the gamma-phosphate. For a significant fraction of the simulation time, forms similar to the alternate structure are observed, and a water molecule is localized near the hydrolytic site. The molecular dynamics simulations of p21-GTP in solution support a postulated hydrolysis mechanism for the biological inactivation of the nucleotide complex based on crystallographic data.

Amino Acid Sequence↗
PubMed1992

Expression of the GTPase activating domain of the neurofibromatosis type 1 (NF1) gene in Escherichia coli and role of the conserved lysine residue.

A small catalytic domain from the neurofibromatosis type 1 gene, NF1-333, consisting of 333 amino acids between residues 1197 and 1528, including an additional N-terminal methionine, was expressed in Escherichia coli as a soluble protein. Its catalytic activity under non-saturating conditions is similar to the full-length p120-GAP but different from truncated GAP-334. Under saturating conditions its kcat and KM are lower. Lys-1422, which is totally conserved in all GAP proteins, was mutated and the properties of the mutant protein investigated. Lys-1422 seems to be essential for the stability of the proteins and not for its catalytic activity.

Amino Acid Sequence↗
PubMed1992

Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product.

The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.

Adenine Nucleotides↗
PubMed1992

Studies on the structure and mechanism of H-ras p21.

Current knowledge of the structure of H-ras p21 is reviewed with particular emphasis on the interaction between guanine nucleotides and the active site of the protein. The nature of the conformational change induced by GTP hydrolysis is discussed. The major change is seen in the region known as the effector loop (loop 2), with significant but less well-defined changes occurring in loop 4, which is implicated in the GTPase reaction. Other evidence concerning the mechanism of GTP hydrolysis and its activation by GAP (GTPase-activating protein) is also discussed. Evidence regarding the rate limiting step in the p21 GTPase reaction, and the manner in which this and possibly other steps are accelerated by GAP, is inconclusive.

Amino Acid Sequence↗
PubMed1992

Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.

The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

Escherichia coli↗
PubMed1992

A universal expression-purification system based on the coiled-coil interaction of myosin heavy chain.

We have constructed a series of Escherichia coli expression vectors that produce high yields of fusion proteins containing the C-terminal fragment of light meromyosin (LMM) from rabbit fast skeletal muscle. The fusion proteins retain the ability of LMM to form polymers in low salt and to be soluble in high salt. Thus they can be easily purified from bacterial extracts with a high salt-low salt extraction procedure and still retain their biochemical properties. We demonstrate the utility of this system for the heterologous production and simple purification of LMM fusions of p21H-ras, the neurofibromatosis type I protein and the Tat and protease proteins of HIV-1.

Amino Acid Sequence↗
PubMed1992

Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity.

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.

Amino Acid Sequence↗
PubMed1992

Differences in GTPase-activating protein activity between liver tumors and normal liver tissue in mice.

The intrinsic GTPase activity of the cellular protein p21ras is strongly increased by two cytosolic proteins, the GTPase-activating protein (GAP) produced by the neurofibromatosis type 1 gene (NF1-GAP) and a GAP of 120 kDa molecular mass (p120-GAP). The GAP-mediated stimulation of p21ras GTPase activity was measured in cytosol obtained from carcinogen-induced liver tumors and normal liver tissues of mice of two strains, namely C3H/He and C57BL/6J. For this purpose, cytosolic extracts were incubated with recombinant human p21ras complexed to [gamma-32P]GTP and the time-dependent decrease in p21ras bound radioactivity was measured. Liver cytosolic extracts mediated an increase in the GTPase activity of wild-type p21ras. There were great differences between tumor and normal tissues in the maximal velocity (Vmax) and in the apparent Michaelis constant (KM) of the p21ras GTPase reaction. Both Vmax and apparent KM were decreased in the liver tumors. Cytosolic extracts isolated from liver tumors that harbored point mutations in codon 61 of the c-H-ras gene did not differ in their activity from extracts obtained from non-mutated liver tumors. Since both GAP proteins are important cellular regulators of the ras signaling pathway and probably also effectors of p21ras, the observed differences in GAP activity may be of relevance for the tumorigenic process in mouse liver.

Animals↗
PubMed1991

Is there a rate-limiting step before GTP cleavage by H-ras p21?

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.

Fluorescent Dyes↗
PubMed1991

p21 with a phenylalanine 28----leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties.

The H-ras gene product p21H has been mutated at Phe-28, which makes a hydrophobic interaction with the guanine base of bound GDP/GTP. The mutation Phe-28----Leu drastically increases nucleotide dissociation rates without affecting association rates. This is due to a perturbed binding of base, alpha- and beta-phosphate, and Mg2+, as evidenced from 31P NMR and fluorescence measurements. The region around the gamma-phosphate appears normal. The affinity of Mg2+ for both the di- and the triphosphate conformation of the mutant was also measured by fluorescence. The association constant is 3.5 x 10(7) M-1 for the Gpp(NH)p complex, 500 times higher than for the GDP form. The mutation does not change appreciably the intrinsic or the GTPase activating protein (GAP)-stimulated GTPase. The mutated protein induces neurite differentiation however when pressure-loaded into PC12 cells, which is equivalent to transformation of NIH 3T3 cells. This shows that p21 (F28L) is converted to the GDP bound form by GAP but is transforming because the high dissociation rate for nucleotides leads to a protein predominantly in the active GTP bound form.

Animals↗
PubMed1991

C-terminal structure and mobility of rabbit skeletal muscle light meromyosin as studied by one- and two-dimensional 1H NMR spectroscopy and X-ray small-angle scattering.

Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy. X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin. The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile. The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other. Essentially the same pattern is observed in intact myosin. In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues. The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5. The pK values of the histidine residues vary between 5.7 and 7.0.

Amino Acid Sequence↗
PubMed1991

The effect of kirromycin on the metal ion coordination in complexes of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

The binding of the antibiotic kirromycin to elongation factor Tu (EF-Tu) leads to a severe line broadening of the Q-band manganese EPR lines of EF-Tu.Mn, EF-Tu.Mn.GDP, as well as EF-Tu.Mn.GDP.Pi indicating that the coordination sphere of the metal ion is changed by this interaction. The number of coordinated water molecules was determined from the 17O-55Mn superhyperfine coupling observable in H2(17)O enriched water; it is pH-dependent in the EF-Tu.Mn.GDP complex, at pH 6.8 probably four water molecules are coordinated with the protein bound manganese, at pH 8.4 one of the water molecules is replaced by a functional group from the protein. Independent of the pH, probably four and five water molecules are bound to the metal ion in the EF-Tu.Mn and in the EF-Tu.Mn.GDP.Pi complex, respectively. Kirromycin does not influence the number of water molecules bound to EF-Tu.Mn.GDP and EF-Tu.Mn.GDP.Pi, but leads to an increase of the number of water molecules coordinated to the metal ion in EF-Tu.Mn. The 17O-55Mn superhyperfine coupling constant in Mn(H2O)6 was determined from the EPR-spectra as 0.24 mT.

Anti-Bacterial Agents↗
PubMed1991

Structural and catalytic properties of a deletion derivative (delta 133-157) of Escherichia coli adenylate kinase.

Escherichia coli adenylate kinase (AKe) as well as the enzyme from yeast and mitochondria differs from the muscle cytosolic variant (AK1) by an insertion of 25 amino acid residues that are missing in AK1. The extra sequence, highly homologous in "large" size variants, is situated between residues 133 and 157 in AKe. Removal of 25 codons in the corresponding adk gene resulted in expression of a modified form of adenylate kinase (delta 133-157 AKe) which still conserved 7% of the maximal activity of the wild-type protein. The apparent Km for nucleotide substrates was increased by a factor of 4.6 (ADP), 23 (ATP) or 43 (AMP) in delta 133-157 AKe when compared with the wild-type enzyme. The secondary structure of delta 133-157 AKe, as well as its thermal stability were very similar to the parent protein. However, the deleted protein was much more sensitive than the wild-type enzyme to inactivation by trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of trypsin digested delta 133-157 AKe revealed accumulation of several well defined fragments which were not observed in the case of wild-type enzyme. We conclude that the additional sequence, although necessary for expression of full activity in AKe, is not critical for catalysis. It is perhaps responsible for interaction of enzyme with other cellular components although a different mechanism of water shielding for large and small size variants of AK can be also envisaged.

Adenylate Kinase↗
PubMed1991

Rabbit skeletal muscle myosin. Unfolded carboxyl-terminus and its role in molecular assembly.

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM-30 and LMM-30C'. LMM-30 consists of 263 amino acids including the original C-terminus of myosin heavy chain. LMM-30C' is colinear with LMM-30, but is devoid of 17 residues at the C-terminus. 1H NMR spectroscopy indicates that the C-terminus of LMM-30, but not of LMM-30C' is unfolded and freely mobile. Furthermore, the present results show that the unfolded C-terminus is essential for molecular assembly of LMM-30; at pH 8.0 LMM-30, but not LMM-30C', formed aggregates upon decreasing the ionic strength.

Amino Acid Sequence↗