High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1. 94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.     

Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes

The mammalian mitochondrial complex consisting of elongation factors EF-Tu and EF-Ts (EF-Tu.Tsmt) is capable of efficiently binding aminoacyl-tRNA to the ribosome in the presence and absence of guanine nucleotides. In the presence of GTP the binding reaction is catalytic. In the absence of guanine nucleotides, or in the presence of a non-hydrolyzable GTP analog, only one round of ribosome binding occurs. EF-Tu.Tsmt is capable of forming a ternary complex with GTP and Escherichia coli Phe-tRNA as demonstrated by gel filtration chromatography, nitrocellulose filter binding, and by protection of the aminoacyl-tRNA bond from hydrolysis. GDP and the non-hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate are also capable of facilitating ternary complex formation with EF-Tu.Tsmt, but are less effective. No kinetic advantage results from the formation of this ternary complex prior to ribosome binding, and EF-Tu.Tsmt may actually bind aminoacyl-tRNA directly to the ribosome prior to binding GTP. These results suggest that a variation of the prokaryotic elongation cycle is occurring in animal mitochondria. N-Ethylmaleimide inhibits the activity of EF-Tu.Tsmt in polymerization and in ribosome binding. However, the activity of the EF-Tsmt which can be measured independently, is not altered.     

Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E. coli EF-Tu

The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E. coli have been investigated. The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E. coli EF-Ts. The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E. coli EF-Ts. The E. coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E. coli EF-Tu:E. coli EF-Ts complex.     

Determination of the kinetics of guanine nucleotide exchange on EF-Tu and EF-Ts: continuing uncertainties

An analysis is made of the rate constants for the reactions involving the interactions of EF-Tu, EF-Ts, GDP, and GTP recently derived by Gromadski et al. [Biochemistry 41 (2002) 162]. Though their measured values appear to allow a reasonable rate of nucleotide exchange sufficient to support rates of protein synthesis in vivo, their data underestimate the thermodynamic barrier involved in nucleotide exchange and therefore cannot be considered definitive. A kinetic scheme consistent with the thermodynamic barrier can be achieved by modification of various rate constants, particularly of those involving the release of EF-Ts from EF-Tu.GTP.EF-Ts, but such constants are markedly different from what are experimentally observed. It thus remains impossible at present satisfactorily to model guanine nucleotide exchange on EF-Tu, catalysed by EF-Ts by a double displacement mechanism, with experimentally derived rate constants. Metabolic control analysis has been applied to determine the degree of flux control of the different steps in the pathway.     

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.     

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.     

Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochondrial cytochrome bc1 complex.

Cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria was crystallized by a batchwise method from protein solution containing sucrose monolaurate using polyethylene glycol-4000 as a precipitant. The red parallelepiped crystals grew to a size of approximately 1 mm x 1 mm x 1 mm. The crystalline protein showed enzymic activity catalyzing electron transfer from ubiquinol-2 to cytochrome c. The subunit composition and absorption spectrum of the crystalline enzyme were identical to those reported previously for the enzyme in solution. The crystal diffracted X-rays to 7.5 A resolution. The diffraction pattern indicated a monoclinic form, space group P2(1), and unit-cell constants of a = 196 A, b = 179 A, c = 253 A and beta = 97 degrees. Most probably four functional units are present in an asymmetric unit.     

Evidence for activities of rabbit reticulocyte elongation factor 1 analogous to bacterial factors EF-Ts and EF-Tu

Preparations have been obtained from rabbit reticulocyte elongation factor 1 (EF-1) that exhibit activities analogous to the heat stable and heat labile factors, EF-Ts and EF-Tu, of $\text{Escherichia coli}$. The heat stable fraction, prepared by heating EF-1 in the presence of GTP, has virtually no activity in poly (U)-directed polyphenylalanine synthesis. The fraction exhibiting activity similar to bacterial EF-Tu is obtained by the interaction of EF-1 with GTP and phenylalanyl-tRNA followed by passage of the solution through a nitrocellulose filter. The filtrate, which alone has low activity in polyphenylalanine synthesis, when combined with the heat stable fraction gives high activity suggesting that the heat stable preparation catalyzes recycling of the filtrate component.     

Crystallization and preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1

Many attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from thermus aquaticus. The crystals belong to hexagonal space group P6(3)(22) and diffract up to 2.6 A. The cell dimensions are a = b = 178 A, c = 238 A and 6 molecules are contained per asymmetric unit.     

Reactivity of essential histidine residues in EF-Tu.GDP and EF-Tu.GTP from Escherichia coli

The microenvironment of histidine residues located in the binding site of elongation factor EF-Tu from Escherichia coli for the 3' terminus of aminoacyl-tRNA is altered during transition of EF-Tu.GDP to EF-Tu.GTP.     

Isolation, crystallization and preliminary X-ray diffraction data of human progastricsin

Human progastricsin, a zymogen of one of the gastric aspartic proteinases, was isolated and crystallized. The crystals belong to the tetragonal space group P4(2)2(1)2, and have unit cell dimensions a = b = 105.5 +/- 0.1 A, c = 70.6 A. The native crystals of progastricsin diffract X-rays at least to 2.5 A and are suitable for a high-resolution X-ray analysis.     

Purification, crystallisation and preliminary X-ray diffraction characterisation of methanol dehydrogenase from Methylosinus trichosporium OB3b

Methanol dehydrogenase was purified from the obligate methanotroph, Methylosinus trichosporium OB3b, in two steps from disrupted biomass by aqueous two-phase partition and ion-exchange chromatography. Copartitioning of a cytochrome c was dependent upon the pH at which aqueous partition was carried out. The native enzyme has a Mr of 120,000, as determined by gel filtration chromatography, and consists of two identical subunits. The purified enzyme contained four electrophoretically distinct isoenzymes, with pI values of 6.3, 6.58, 6.63 and 6.88. The native enzyme has been crystallised in a form suitable for high-resolution X-ray crystallographic studies. The crystals diffract to better than 0.19 nm spacing and are relatively stable to irradiation with X-rays. The space group is P6(1)22 (or P6(5)22) with cell dimensions a = b = 10.21 nm, c = 29.32 nm and the crystal probably contains a single monomer in the asymmetric unit.     

Analysis of rate constants governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The kinetics of the heterologous exchange of GDP bound to EF-Tu by free GTP catalysed by EF-Ts have been analysed with a view to correlating results obtainable with different computational procedures. The affinity of EF-Ts for EF-Tu.GTP was found to be somewhat less than previously proposed by Romero et al. (Biochemistry 260, 6167:1985) though still greater than for EF-Tu.GDP. There is a close interrelationship between the constants for the binding of GTP to EF-Tu.EF-Ts and of EF-Ts to EF-Tu.GTP. The declining fractional rate of exchange observed by Romero et al. during displacement of GDP by GTP appears to be dependent on the ratio of the rate constants (k-1 + k-2)k4/k1k-2 as defined in the text, not on that of K4/K1 as they proposed.     

Crystallization and preliminary X-ray diffraction studies of antigen--antibody complexes

Monoclonal antibodies of predefined specificity have been purified and crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab fragments separately. Intrasegmental mobility in Fabs has rarely been an obstacle to their crystallization. The immune system, however, provides a large functional and structural diversity of antibody molecules suitable for crystallization and X-ray diffraction studies.     

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome.     

Crystallization of and preliminary X-ray data for bovine carbonic anhydrase III

Crystals of bovine carbonic anhydrase III have been grown in a solution of polyethylene glycol. The crystals are monoclinic, space group P2(1), with the unit cell parameters a = 50.6 A, b = 44.7 A, c = 56.9 A, and beta = 90.3 degrees. The asymmetric unit contains 1 molecule. The diffraction pattern extends beyond 2.0-A resolution.     

Crystallization and preliminary X-ray diffraction studies of bovine recombinant immune interferon

Reproducible conditions have been established for the crystallization of recombinant bovine immune interferon. Two cystalline forms of this protein were obtained. A tetragonal form, space group P422, with unit cell dimensions a = b = 59.0 A and c = 125.7 A and an orthorhombic form, space group P2(1)2(1)2(1), with unit cell dimensions a = 42.80 A, b = 79.90 A and c = 85.64 A were obtained under similar crystallization conditions. The orthorhombic form diffracts to 2.6 A resolution, contains a single interferon dimer in the asymmetric unit of structure and is suitable for X-ray diffraction analysis.