Parasite plastids: maintenance and functions.

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima.

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.

Interaction between Ran and Mog1 is required for efficient nuclear protein import.

Mog1 is a nuclear protein that interacts with Ran, the Ras family GTPase that confers directionality to nuclear import and export pathways. Deletion of MOG1 in Saccharomyces cerevisiae (Deltamog1) causes temperature-sensitive growth and defects in nuclear protein import. Mog1 has previously been shown to stimulate GTP release from Ran and we demonstrate here that addition of Mog1 to either Ran-GTP or Ran-GDP results in nucleotide release and formation of a stable complex between Mog1 and nucleotide-free Ran. Moreover, MOG1 shows synthetic lethality with PRP20, the Ran guanine nucleotide exchange factor (RanGEF) that also binds nucleotide-free Ran. To probe the functional role of the Mog1-Ran interaction, we engineered mutants of yeast Mog1 and Ran that specifically disrupt their interaction both in vitro and in vivo. These mutants indicate that the interaction interface involves conserved Mog1p residues Asp(62) and Glu(65), and residue Lys(136) in yeast Ran. Mutations at these residues decrease the ability of Mog1 to bind and release nucleotide from Ran. Furthermore, the E65K-Mog1 and K136E-Ran mutations in yeast cause temperature sensitivity and mislocalization of a nuclear import reporter protein, similar to the phenotype observed for the Deltamog1 strain. Our results indicate that a primary function of Mog1 requires binding to Ran and that the Mog1-Ran interaction is necessary for efficient nuclear protein import in vivo.

Chemistry of sulforhodamine--amine conjugates.

Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The interaction of each isomer of the latter conjugates with myosin subfragment 1 has been characterized. Significant differences between the two isomers are observed in these interactions.

Mechanism of nucleotide release from Rho by the GDP dissociation stimulator protein.

Guanine nucleotide dissociation stimulator (GDS) promotes the release of tightly bound GDP from various Ras superfamily proteins, including RhoA, Rac1, K-Ras, Rap1A, and Rap1B. It displays no significant sequence homology to other known exchange factors for small G-proteins. Studies are reported here of the mechanism of GDS-mediated nucleotide release from RhoA using a combination of equilibrium and stopped-flow kinetic measurements, employing fluorescent N-methylanthraniloyl (mant) derivatives of GDP and 2'-deoxyGDP. It is proposed that GDS operates by an associative displacement mechanism where stimulated nucleotide release from the Rho.mantGDP complex occurs via a transiently populated ternary complex (Rho.GDS.mantGDP). In kinetic experiments where excess GDS was mixed with the Rho.mantGDP complex, stimulated mantGDP dissociation rates of 1 s(-)(1) were measured during a single turnover, representing a 5000-fold enhancement over the intrinsic rate of mantGDP dissociation from Rho. The stable, nucleotide-free binary complex Rho.GDS was isolated. When the Rho.GDS complex was mixed with excess mantGDP, a biphasic increase in fluorescence occurred, the observed rate constants of which both reached saturating values at high mantGDP concentrations. This is compelling evidence that an isomerization of the Rho.GDS.mantGDP ternary complex is an important feature of the mechanism of nucleotide release.

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

Distances between DNA and ATP binding sites in the TyrR-DNA complex.

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It binds to DNA as a homodimer with a subunit molecular mass of 57 640 Da, each of which has a single site for the binding of ATP within a central structural domain. This paper reports distances between four sites on the DNA and the ATP binding site as determined by fluorescence resonance energy transfer. The DNA was a 30mer containing a centrally located binding site for TyrR. Replacement of a thymidine residue with an aminouridine residue at positions -9, -7, -3, and 2 of the palindromic oligonucleotide sequence enabled the placement of a single fluorescein group along the major groove of the DNA. The energy transfer acceptor was ATP labeled with a rhodamine group through positions 2' and 3' of the ribose, positions that are known to cause minimal interference with the binding of ATP to protein. The dissociation constant for the binding of rhodamine-ATP to TyrR was 300 nM as determined by steady-state fluorescence anisotropy titrations. The energy transfer efficiencies were determined by measuring the level of quenching of donor fluorescence on binding rhodamine-ATP to the TyrR-DNA complex. The experimental transfer efficiencies were compared to theoretical values calculated for a model of the DNA-TyrR complex in which the position of the ATP binding site was allowed to vary over the surface of the monomer unit. Theory was written to account for the transfer from one donor to two acceptors, one on each monomer unit of the TyrR dimer. The results indicate that the ATP binding site is about 40-45 A from the nearest point on the DNA and distant from the DNA helix-turn-helix binding domain. The effects of ATP binding of (i) increasing the TyrR binding affinity by a factor of 4-5 and (ii) permitting the binding of the tyrosine corepressor must therefore occur because of a significant allosteric change in the conformation of the protein.

Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma.

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.

Correlation between self-association modes and GTPase activation of dynamin.

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 x 10(17) M(-3). Stopped-flow fluorescence experiments show that the association rate constant for 2'(3')-O-N-methylanthraniloyl (mant)GTP is 7.0 x 10(-5) M(-1) s(-1) and the dissociation rate constant is 2.1 s(-1), whereas the dissociation rate constant for mantdeoxyGDP is 93 s(-1). We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride.

The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.

The importance of two conserved arginine residues for catalysis by the ras GTPase-activating protein, neurofibromin.

Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 10(5)-fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras.GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate contributions of each of these residues to catalysis and binding affinity to Ras were measured. Both the R1276A and the R1391A mutant NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured at saturating concentrations. In contrast, there was only a minor effect of either mutation on NF1-334 affinity for wild-type Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely essential, because the mutant NF1-334 proteins increase the intrinsic Ras.GTPase by at least 100-fold. The roles of Arg1276 and Arg1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with Ras.

Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase.

The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations. An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study. Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate. The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting. Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively. Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase.

Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue.

Small G proteins of the Rho family, which includes Rho, Rac and Cdc42Hs, regulate phosphorylation pathways that control a range of biological functions including cytoskeleton formation and cell proliferation. They operate as molecular switches, cycling between the biologically active GTP-bound form and the inactive GDP-bound state. Their rate of hydrolysis of GTP to GDP by virtue of their intrinsic GTPase activity is slow, but can be accelerated by up to 10(5)-fold through interaction with rhoGAP, a GTPase-activating protein that stimulates Rho-family proteins. As such, rhoGAP plays a crucial role in regulating Rho-mediated signalling pathways. Here we report the crystal structure of RhoA and rhoGAP complexed with the transition-state analogue GDP.AlF4- at 1.65 A resolution. There is a rotation of 20 degrees between the Rho and rhoGAP proteins in this complex when compared with the ground-state complex Cdc42Hs.GMPPNP/rhoGAP, in which Cdc42Hs is bound to the non-hydrolysable GTP analogue GMPPNP. Consequently, in the transition state complex but not in the ground state, the rhoGAP domain contributes a residue, Arg85(GAP) directly into the active site of the G protein. We propose that this residue acts to stabilize the transition state of the GTPase reaction. RhoGAP also appears to function by stabilizing several regions of RhoA that are important in signalling the hydrolysis of GTP.

Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP.

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signalling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 10(5) times. We report here the crystal structure of Cdc42Hs, with the non-hydrolysable GTP analogue GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7A resolution. In the complex Cdc42Hs interacts, mainly through its switch I and II regions, with a shallow pocket on rhoGAP which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit G(i alpha1), we propose that it adopts a different conformation during the catalytic cycle which enables it to stabilize the transition state of the GTP-hydrolysis reaction.

Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum.

Small GTPases interact with a variety of proteins that affect nucleotide binding and cleavage. GTPase activating proteins (GAPs) are one class of these proteins that act by accelerating the intrinsic GTPase rate resulting in the formation of the biologically inactive GDP-bound form of the GTPase. For the Rho subfamily of GTPases, there is a growing number of proteins with rhoGAP activity that are identifiable by a homologous region of about 150 amino acids. We have exploited this homology using the polymerase chain reaction to clone the first rhoGAP homolog, called DdRacGAP, from the slime mold Dictyostelium discoideum. The GAP domain of DdRacGAP (amino acids 1-212), when expressed and purified from Escherichia coli, is active on both Dictyostelium and human Rho family GTPases but not human Ras. The full-length protein is 1356 amino acids in length and has several interesting homologies in addition to the GAP domain, including an SH3 domain, a dbl homology domain, and a pleckstrin homology domain.