Proton NMR studies of the GDP.Mg2+ complex of the Ha-ras oncogene product p21

Two-dimensional proton NMR studies were performed on the c-Ha-ras encoded proto-oncogene product p21C. COSY and NOESY spectra of the p21C.GDP.Mg2+ complex show that the ribose H1 proton of the bound GDP is in close proximity to the aromatic side chain of a phenylalanyl residue. From sequence homology with the bacterial elongation factor Tu (EF-Tu) and the known X-ray structure of the EF-Tu.GDP.Mg2+ complex it may be inferred that the Phe residue in question is either Phe78 or Phe82 in the p21 sequence.     

Kinetics of interaction of nucleotides with nucleotide-free H-ras p21

A method is described for the convenient preparation of substantial quantities of nucleotide-free p21 or of 1:1 complexes with nucleotides other than GDP. The nucleotide-free protein has been used for kinetic studies of the binding of GDP and GTP, making use of the fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP and -GTP. Stopped-flow studies have led to the formulation of a two-step binding mechanism for both GDP and GTP, involving initial rapid but weak binding of the nucleotide followed by a relatively slow (10-20 s-1 at 25 degrees C; 3-5 s-1 at 5 degrees C) quasi-irreversible isomerization reaction. By use of a nonequilibrium competition method, guanosine and GMP have been shown to interact weakly but significantly with p21 (dissociation constants of 153 and 29 microM, respectively). The presence of guanosine or GMP at the active site of p21 leads to a marked stabilization of p21 against spontaneous denaturation when compared with the nucleotide- and nucleoside-free protein.     

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.     

Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules

The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12----Arg, Gly-12----Val, Gln-61----His, Gln-61----Leu, which are all oncogenic, and the effector region mutant Asp-38----Glu. The resolutions of the crystal structures range from 2.0 to 2.6 A. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the gamma-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.     

Proton NMR studies of vesicles incorporating glycophorin

The effects of incorporation of glycophorin. the major sialoglycoprotein or the human crythrocyte membrane, on the lipid of small vesicles have been studied using proton NMR and electron microscopy. In contrast to the incorporation of other peptides, the major effect is apparently the clustering of vesicles without fusion. The relative mobility of lipids of the vesicle. monitored by changes in proton spin-lattice time, is only moderately effected by the presence of protein. The methylene protons of the lipid chains are subject to a somewhat greater restriction of motion following the incorporation of glycophorin than are the protons of the head group.     

Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products

We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins. It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein. We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins [McCormick, F., Clark, B. F. C., LaCour, T. F. M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J. (1985) Science (Washington, D.C.) 230, 78-82]. Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core. Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.     

Proton NMR studies of myohemerythrin from Themiste zostericola

 One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin (Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the differentiation of the signals from the N_δ-H protons of the five N_ε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr, is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α, β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the binding of N₃ ^– to one of the Fe(II) sites in deoxyMHr, the overlapping His N_δ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in the ¹H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24. Received: 20 May 1996 / Accepted: 24 October 1996     

Characterisation of the metal-ion-GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O-Mn superhyperfine coupling and by kinetic methods

Kinetic studies on the interaction of three Ha-ras-encoded p21 proteins with GDP and MgGDP have yielded values for the association (10(6)-10(7) M-1 s-1) and dissociation (10(-3)-10(-5) s-1) rate constants at 0 degrees C. Dramatic differences in the rate constants were not observed for the three proteins. Under non-physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t, but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7 X 10(10) M-1 for p21c to 1.3 X 10(11) M-1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O-labelled GDP showed direct coordination of a beta-phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16-0.22 mT, but no interaction with the alpha-phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotides was estimated to be greater than 10(5) M-1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the alpha-phosphate group. These results have been used to construct a model for the interactions of Mg X GDP with the active site of p21 proteins.     

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.     

Structural basis of p21H-ras molecular switch inhibition by a neutralizing antibody

The ras oncogene product p21 functions as a molecular switch in the early section of the signal transduction pathway that is involved in cell growth and differentiation. When the protein is in its GTP-complexed form it is active in signal transduction, whereas it is inactive in its GDP-complexed form. The transforming activity of p21^ras is neutralized by the mouse monoclonal antibody Y13-259, possibly by preventing GDP-GTP exchange. A molecular model of the variable fragment of Y13-259 has been derived using a knowledge-based prediction approach and computer-assisted modeling techniques. An analysis of this model while complexed with p21^ras/(GDP) indicated that the two molecular switch regions are constrained by complex formation. Antibody binding inhibits GDP-GTP exchange through a mechanism of steric hindrance. Having identified necessary bound sites for inhibition, and explored their electrostatic properties, it should be possible to proceed with the design of antibody mimics as therapeutic agents in cancer control.     

Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions

The protein farnesyltransferase purified from rat brain contains two nonidentical subunits, alpha and beta. The holoenzyme forms a stable complex with [3H]farnesyl pyrophosphate (FPP) that can be isolated by gel filtration. The [3H]FPP is not covalently bound to the enzyme; it is released unaltered when the enzyme is denatured. When incubated with an acceptor such as p21H-ras, the complex transfers [3H]farnesyl from the bound [3H]FPP to the ras protein. This transfer is not sensitive to dilution by unbound FPP, suggesting that the [3H]FPP is bound at a site that leads to direct transfer to the p21H-ras acceptor. Cross-linking studies show that the p21H-ras binds to the lower molecular weight subunit (beta-subunit), raising the possibility that the [3H]FPP binds to the alpha-subunit. If this suggestion can be confirmed, it would invoke a reaction mechanism in which the alpha-subunit acts as a prenyl pyrophosphate carrier that delivers FPP to p21H-ras which is bound to the beta-subunit.     

Proton correlation NMR studies of metabolism in Rhodopseudomonas palustris

A 1H correlation NMR study is reported, on the metabolism of a photosynthetic bacterium, Rhodopseudomonas palustris, in dark and light anaerobic conditions. Alkali treatment as well as sonication of the cells were employed to follow the process of accumulation and decomposition of poly-beta-hydroxybutyrate (PHB) which is the reserve material for the bacterium. It was shown that synthesis of PHB from trans-crotonate proceeds in the granules of the cells. It was also demonstrated that under anaerobic light conditions photometabolism and glycolysis generally compete with concomitant synthesis and decomposition of PHB, respectively, and that glycolysis gradually replaces photometabolism with aging of the cells. In contrast, glycolysis is always predominant in the dark and PHB is primarily used as the carbon source. It was observed that photo-induced transport of beta-hydroxybutyrate through the membrane occurs when photometabolism and glycolysis are equally active in the light. The implications of this observation are briefly discussed.     

Proton NMR spectroscopic studies of dipeptidase in human erythrocytes

In studies on human erythrocyte metabolism in situ, high resolution (400 MHz)¹H spin-echo NMR spectroscopy was used to follow the time dependence of hydrolysis of glycylglycine and L-cysteinylglycine in intact cells and their lysates. The concentration dependence of the hydrolysis of L-cysteinylglycine was described by a rectangular hyperbola with K_m, 3.5 ± 0.6 mmol/llysate and V_max, 64.2 ± 3.2 mmol/llysate/h. We demonstrated that glycylglycine readily enters the erythrocyte and we introduce a means of analysing the data from the coupled reaction sequence; the sequence consists of transport followed by enzyme catalysed hydrolysis.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.     

Proton NMR studies on soya bean lipoxygenase-1.

The 270 MHz 1H-NMR spectrum of soya bean lipoxygenase-1 (linoleate: oxygen oxidoreductase, EC 1.13.11.12) was investigated at 298 K and at several pH values. A large fraction of the protein (50%) was found to be effectively random coil.     

Proton NMR studies of two helices in tuna ferricytochrome c

1H Nuclear magnetic resonance assignments are given for the NH and C alpha H protons of two alpha-helical segments of tuna ferricytochrome c. The assignments were obtained using two-dimensional nuclear magnetic resonance sequential assignment procedures and illustrate the applicability of these methods to medium-sized proteins. By comparing nuclear Overhauser intensities between the NH and C alpha H protons the precise structures of the two helical segments are compared and their deviations from ideality are discussed.     

Proton NMR studies of angiotensin II and its analogs in aqueous solution

The¹H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H₂O/10% D₂O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population belowpH 5.5 and increases to 12–20% aroundpH 7.0. The two sets of signals result from acis-trans isomerization of the His-Pro peptide bond with the major resonances arising from thetrans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants J_NH-CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY andpH-titration experiments. Moreover,pH effects and the unusual amide exchange behavior of the Arg NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH₂ protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteinsPreliminary results of this work have been presented at the XIIth International Conference on Magnetic Resonance in Biological Systems in abstract form (1988).