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I Schlichting

Publications and source records attributed to I Schlichting.

51 records · Page 3Linked to original sources
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↗
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↗
PubMed1990

Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases.

A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand alpha,omega-di[(3' or 2')-O-(N-methylanthraniloyl)adenosine-5'] pentaphosphate (mAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 x 10(-9) to 5 x 10(-2) M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. The techniques are used to describe the properties of a mutant enzyme with Gln-28----His (Q28H) prepared by site-directed mutagenesis in comparison to those of wild-type AK from Escherichia coli. This newly introduced histidine is already present in most other adenylate kinases and was regarded to be important or even essential for the catalytic reaction of AK. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. However, steady-state catalytic studies and binding experiments showed that the affinities for substrates and inhibitors are elevated from 3-fold (AMP) to 5-fold (ATP) to 15-fold (AP5A) compared to those of the wild-type enzyme. Together with the results obtained by Tian et al. [Tian, G., Sanders, C. R., Kishi, F., Nakazawa, A., & Tsai, M.-D. (1988) Biochemistry 27, 5544-5552] on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.

Adenylate Kinase↗
PubMed1990

Structurally and catalytically important residues in the phosphate binding loop of adenylate kinase of Escherichia coli.

Amino acids in the phosphate binding loop of adenylate kinase of Escherichia coli were mutated by site-directed mutagenesis. The mutant proteins with a Pro-9----Gly (P9G) and with a Lys-13----Gln (K13Q) exchange were overexpressed and purified. They were characterized by steady-state kinetics, fluorescence binding, and structural studies, together with the phosphate binding loop mutants P9L and G10V prepared earlier [Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720]. The results obtained show that all these mutations change the structure of the protein as evidenced by NMR spectroscopy and temperature-stability studies. All the mutant proteins have increased dissociation constants for substrates and inhibitors, but their catalytic activity, except for K13Q, is not reduced. The results obtained with K13Q suggest that this lysine residue, which is conserved in all guanine and many adenine nucleotide proteins, might have an important role in catalysis.

Adenylate Kinase↗
PubMed1990

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.

Adenosine Triphosphate↗
PubMed1990

Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis.

Crystals of Ha-Ras p21 with caged GTP at the active site have been used to investigate the conformational changes of p21 on GTP hydrolysis. The structure of the short-lived p21.GTP complex was determined by Laue diffraction methods. After GTP hydrolysis, substantial structural changes occur in the parts of the molecule implicated in the interaction with GTPase-activating protein. The trigger for this process seems to be a change in coordination of the active-site Mg2+ ion as a result of loss of the gamma-phosphate of GTP.

Binding Sites↗
PubMed1990

Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli.

The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.

Animals↗
PubMed1990

Proton NMR studies of transforming and nontransforming H-ras p21 mutants.

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.

Base Sequence↗
PubMed1989

C-terminal truncation of p21H preserves crucial kinetic and structural properties.

The human c-Ha-ras protooncogene product p21C was truncated at the C terminus by 23 amino acids. The resulting G-binding domain, p21 (1-166) = p21C', can be crystallized as a complex with the slowly hydrolyzing GTP analogues guanosin-5'-[beta,gamma-imido]triphosphate, guanosin-5'-[beta,gamma-methylene]triphosphate, and guanosin-5'-O-(3-thiotriphosphate). We show here that this protein has biochemical properties very similar to those of the intact protein. Activating mutations in position 12 (Gly12----Val; Gly12----Arg) have the same effect on the properties of the truncated protein as on intact protein. Nuclear magnetic resonance (NMR) measurements show no apparent effect of the C-terminal deletion on the solution structure of p21. This suggests that neither the structure of the G-binding domain nor any of its biochemical properties are markedly influenced by the truncation.

Amino Acid Sequence↗
PubMed1989

Biochemical and crystallographic characterization of a complex of c-Ha-ras p21 and caged GTP with flash photolysis.

The GTP binding domain of the c-Ha-ras protooncogene product (p21'c) and the corresponding region from an oncogenic mutant form of the protein in which glycine at position 12 has been replaced by valine [p21'(G12V)] have been crystallized with P3-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate (caged GTP) at their active sites. The crystals give x-ray diffraction patterns to a resolution of better than 0.3 nm. Photolysis can be achieved in the crystal, after which GTP hydrolysis takes place at the rate expected from solution studies. Complete x-ray data sets have been obtained for the starting caged-GTP state and the final GDP state after photolysis and hydrolysis, demonstrating the feasibility of time-resolved structural investigations of the process of GTP hydrolysis.

Binding Sites↗
PubMed1988

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.

Guanine Nucleotides↗
PubMed1986

31P-NMR spectra of the Ha-ras p21.nucleotide complexes.

Phosphorus nuclear magnetic resonance spectra of the Ha-ras oncogene product p21 and its nucleotide complexes have been obtained. It is shown that the 31P nuclear magnetic resonance spectra of a number of nucleotide-enzyme complexes show some common features. In particular, the chemical shift values of the beta-phosphorus resonance of enzyme-bound NTP and NDP (N = A, G) of hydrolases exhibit a downfield shift virtually identical for myosin, elongation factor Tu, and the Ha-ras oncogene product p21. This suggests that the stereochemistry around the beta-phosphorus might be similar in these compounds.

Guanosine Diphosphate↗