Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.
SummaryA soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O 2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox þ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O 2 . Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O 2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O 2 in the regulation of pneumococcal transformability and virulence.
Regulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.
of H-Ras bound to GDP (Milburn et al., 1990) and to the Gé rard Le Bras, Gisè le Le Bras, non-hydrolysable GTP analogues GPPNP (Pai et al., Isabelle Janoueix-Lerosey 2 , 1990) and GPPCP (Milburn et al., 1990) (Willumsen et al., 1986), and the recent crystal structure interactions (Nassar et al., 1995(Nassar et al., , 1996. This region probably constitutes a general docking site for the effectors The small G protein Rap2A has been crystallized in of small G proteins. The switch II region, on the other complex with GDP, GTP and GTPγS. The Rap2A-hand, is not essential for proper effector recognition, but GTP complex is the first structure of a small G protein may interact with guanine nucleotide exchange factors with its natural ligand GTP. It shows that the hydroxyl (reviewed in Polakis and McCormick, 1993). As most of group of Tyr32 forms a hydrogen bond with the these data were obtained for H-Ras, it remains essentially γ-phosphate of GTP and with Gly13. This interaction unsettled whether the numerous small G proteins of does not exist in the Rap2A-GTPγS complex. Tyr32 is the Ras family undergo the same GDP/GTP structural conserved in many small G proteins, which probably transition as H-Ras . also form this hydrogen bond with GTP. In addition,The H-Ras structures, by locating essential proteinTyr32 is structurally equivalent to a conserved arginine nucleotide interactions, have also been a reference in the that binds GTP in trimeric G proteins. The actual longstanding debate on how the GTPase activity of small participation of Tyr32 in GTP hydrolysis is not yet G proteins works (reviewed in Maegley et al., 1996). The clear, but several possible roles are discussed. The rate of hydrolysis of GTP is important for the duration of conformational changes between the GDP and GTP the association of the G protein with its GTP-specific complexes are located essentially in the switch I and partners, and it is usually very low (compiled in Zerial II regions as described for the related oncoprotein and Huber, 1995). The crystal structures of H-Ras define H-Ras. However, the mobile segments vary in length candidate residues for activation of the water molecule that and in the amplitude of movement. This suggests that attacks the γ-phosphate of GTP, and for the stabilization of even though similar regions might be involved in the the transition state of the GTPase reaction. It is not known, GDP-GTP cycle of small G proteins, the details of the however, how the GTPase reaction is designed to act as changes will be different for each G protein and will a timer and/or wait for interactions triggered by GTPaseensure the specificity of its interaction with a given set activating proteins (GAPs). of cellular proteins.Rap proteins, which include Rap1A, Rap1B, Rap2A Keywords: crystal structure/G proteins/GTP hydrolysis/ and Rap2B, have~50% sequence identity with Ras proteins Rap/Ras (reviewed in Bokoch, 1993). Rap1 was independently cloned by sequence homology (Pizon et al., 1988) and by its ability to revert the transformed ...
Selective hsp90 inhibitors simultaneously destabilize and deplete key signaling proteins involved in cell proliferation and survival, angiogenesis, and metastasis. Investigation of novobiocin analogues lacking the noviose moiety as novel inhibitors of hsp90 was carried out. A novel series of 3-aminocoumarin analogues has been produced and screened in cell proliferation, and the molecular signature of hsp90 inhibition was assessed by depletion of estrogen receptor, HER2, Raf-1, and cdk4 in human breast cancer cells. This structure-activity relationship study highlights the crucial role of the C-4 and/or C-7 positions of coumarin which appeared to be essential for degradation of hsp90 client proteins. Removal of the noviose moiety in novobiocin together with introduction of a tosyl substituent at C-4 or C-7 coumarins provides 6e and 6f as lead structures which compared favorably with novobiocin as demonstrated by enhanced rates of cell death. The processing and activation of caspases 7 and 8 and the subsequent cleavage of PARP by 6e suggest stimulation of the extrinsic apoptosis pathway.
The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense coon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.The thrA gene is the first structural gene of the threonine operon of Escherichia coil K-12 (1, 2). It is composed of two parts, thrAl and thrA2 and codes for a bifunctional enzyme, aspartokinase I-homoserine dehydrbgenase I (EC 2.7.2.4 and EC 1.1.1.3). The native enzyme (3) is a tetramer with each chain carrying, on discrete domains, the aspartokinase I and homoserine dehydrogenase I activities, which are regulated allosterically by L-threonine. Limited proteolysis of the native enzyme leads to a homodimeric fragment having the same COOHterminal sequence as the native enzyme having only the dehydrogenase activity and no longer inhibited by threonine (3). On the other hand, a polypeptide chain synthesized by an ochre mutant that has the same NH2 terminus as the native enzyme assembles as a tetramer having only the aspartokinase activity, still regulated by threonine (3). The determination of the primary structure of aspartokinase I homoserine-dehydrogenase I seemed warranted for a number of reasons. Sequence information was important to understand enzyme structure-function relationships and to elucidate the allosteric properties of the enzyme. It should permit the study of possible evolutionary relationships between the different proteins coded by the threonine operon and the homology with the isofunctional enzymes in E. coli, aspartokinase II-homoserine dehydrogenase II, coded by metL, and aspartokinase III coded by lysC.
Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6-phosphate into fructose 1,6-bisphosphate in Lactobacillus buigaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35-kDa subunits. Its N-terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearotherrnophiius, suggesting that the three proteins have closely related structures.The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6-phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6-phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenoipyruvate. Partial inhibition by phosphoenoipyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls V,,,. Protection against thermal denaturation shows that the enzyme binds phosphoenofpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R-like conformation since they bind fructose 6-phosphate, whereas the regulatory sites have a T-like conformation since they bind phosphoenolpyruvate and not GDP.L . bulgaricus is one of the bacteria used by the food industry for large-scale fermentation of milk into cheese and yoghurt, but despite its economic importance, little is known of its biochemistry. L. bulgaricus has a simplified catabolism: (a) it can utilize only three sugars (glucose, fructose and lactose) as a carbon-energy source, (b) it is a strict fermenting bacterium which lacks a respiratory chain, (c) its fermentation produces almost exclusively lactic acid [ 11. L . buigaricus appears then to produce most (if not all) of the energy required for its growth solely by the glycolytic breakdown of sugars.In many organisms, the flux through the glycolytic pathway is, at least in part, controlled at the level of the reaction:ATP +fructose 6-phosphate --f ADP + fructose 1,B-bisphosphate, catalyzed by phosphofructokinase. Phosphofructokinases from a variety of sources are indeed endowed with rather sophisticated regulatory properties, with a marked cooperative behavior and/or potent allosteric effectors [2]. Also, one form of this enzyme appears to be rather conserved
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