Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP.

In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate. In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24). The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack. This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).

X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain.

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.

X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with dinitrophenylaminoethyl-, dinitrophenylaminopropyl-, o-nitrophenylaminoethyl-, m-nitrophenylaminoethyl-, p-nitrophenylaminoethyl-, and o-nitrophenyl-N-methyl-aminoethyl-diphosphate.beryllium fluoride have been determined to better than 2.3-A resolution. The structure of the protein and nucleotide binding pocket in these complexes is very similar to that of S1dC.ADP.BeF(x) (Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). The position of the triphosphate-like moiety is essentially identical in all complexes. Furthermore, the alkyl-amino group plays the same role as the ribose by linking the triphosphate to the adenine binding pocket; however, none of the phenyl groups lie in the same position as adenine in S1dC.MgADP.BeF(x), even though several of these nucleotide analogs are functionally equivalent to ATP. Rather the former location of adenine is occupied by water in the nanolog complexes, and the phenyl groups are organized in a manner that attempts to optimize their hydrogen bonding interactions with this constellation of solvent molecules. A comparison of the kinetic and structural properties of the nanologs relative to ATP suggests that the ability of a substrate to sustain tension and to generate movement correlates with a well defined interaction with the active site water structure observed in S1dC.MgADP.BeF(x).

X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPgammaS, and MgADP are reported at 2.1, 1.9, and 2.1 A resolution, respectively. Crystals were obtained by cocrystallization and were isomorphous with respect to those of S1dC. MgADP.BeFx [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972]. In all three structures, the electron density for the entire nucleotide was clearly discernible. The overall structures of all three complexes are very similar to that of the beryllium fluoride complex which suggests that the differences in the physiological effects of ATPgammaS and AMPPNP are due to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower affinity of AMPPNP for myosin. In S1dC.MgAMPPNP, the presence of the bridging nitrogen prompts the side chain of Asn233 to rotate which disrupts the hydrogen bonding pattern in the nucleotide binding pocket and alters the water structure surrounding the ribose hydroxyl groups. It appears that this change is responsible for the reduced affinity of AMPPNP for myosin relative to ATPgammaS. In contrast to the G-proteins, there is no major change in the conformation of the ligands that coordinate the nucleotide in S1dC.MgADP. This is due to three water molecules that adopt the approximate positions of the three oxygens on the gamma-phosphate and maintain the interactions with the Mg2+ ion and protein molecule. Interestingly, the thiophosphate group is evident in S1dC.MgATPgammaS even though it is slowly hydrolyzed by myosin. This suggests that the conformation observed here and in chicken skeletal myosin subfragment-1 [Rayment, I., et al. (1993) Science 261, 50-58] is unable to hydrolyze ATP and represents the structure of the prehydrolysis weak binding state of myosin.

X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.

Metal ion separations in polyethylene glycol-based aqueous biphasic systems: correlation of partitioning behavior with available thermodynamic hydration data.

Solvent extraction utilizing an oil-water mixture (e.g., chloroform-water) and a suitable complexant, is a proven technology for the selective removal and recovery of metal ions from aqueous solutions. Aqueous biphasic systems (ABS), formed by mixing certain inorganic salts and water-soluble polymers, or by mixing two dissimilar water-soluble polymers, have been studied for more than 40 years for the gentle, non-denaturing separation of fragile biomolecules, yet ABS have been virtually ignored as a possible extraction technology for metal ions. In this report we review our metal ion partitioning work and discuss the three major types of partitioning: (1) those rare instances that the metal ion species present in a given solution partitions to the PEG-rich phase without an extractant; (2) the use of halide salts which produce a metal anion complex that partitions to the PEG-rich phase; and (3) the use of a water-soluble extractant which distributes to the PEG-rich phase. In addition, we correlate the partitioning behavior we observed with available thermodynamic data for metal ions and their complexes.

Partitioning behavior of group 1 and 2 cations in poly(ethylene glycol)-based aqueous biphasic systems.

The partitioning behavior of several Group 1 and 2 cations was investigated in poly(ethylene glycol) (PEG)-based aqueous biphasic systems. All of these metal ions prefer the salt-rich phase over the PEG-rich phase with distribution ratios all well below one regardless of the system investigated. The relative salting-out ability of the individual cations can be directly correlated to their Gibbs free energy of hydration (delta G(hyd)). In addition, the relative magnitude of the distribution ratios for these metal ions can also be explained in terms of delta G(hyd).

A controlled trial of intravenous immune globulin to reduce nosocomial infections in very-low-birth-weight infants. National Institute of Child Health and Human Development Neonatal Research Network.

BACKGROUND: Nosocomial infections are a major cause of morbidity and mortality in premature infants. As a rule, their low serum gamma globulin levels at birth subsequently decline to hypogammaglobulinemic values; hence, prophylactic administration of intravenous immune globulin may reduce the rate of hospital-acquired infections. METHODS: In this prospective, multicenter, two-phase controlled trial, 2416 infants were stratified according to birth weight (501 to 1000 g and 1001 to 1500 g) and randomly assigned to an intravenous immune globulin group (n = 1204) or a control group (n = 1212). Control infants were given placebo infusions during phase 1 of the study (n = 623) but were not given any infusions during phase 2 (n = 589). Infants weighing 501 to 1000 g at birth were given 900 mg of immune globulin per kilogram of body weight, and infants weighing 1001 to 1500 g at birth were given a dose of 700 mg per kilogram. The immune globulin infusions were repeated every 14 days until the infants weighed 1800 g, were transferred to another center, died, or were sent home from the hospital. RESULTS: Nosocomial infections of the blood, meninges, or urinary tract occurred in 439 of the 2416 infants (18.2 percent): 208 (17.3 percent) in the immune globulin group and 231 (19.1 percent) in the control group (relative risk, 0.91; 95 percent confidence interval, 0.77 to 1.08). Septicemia occurred in 15.5 percent of the immune globulin recipients and 17.2 percent of the controls. During phase 1 the rate of nosocomial infections was 13.4 percent in the immune globulin group and 17.8 percent in the control group; the respective rates during phase 2 were 21.0 percent and 20.4 percent. The predominant organisms included gram-positive cocci (53.0 percent), gram-negative bacilli (22.4 percent), and candida species (16.0 percent). Adverse reactions were rarely observed during the infusions. Immune globulin therapy had no effect on respiratory distress syndrome, bronchopulmonary dysplasia, intracranial hemorrhage, the duration of hospitalization, or mortality. The incidence of necrotizing enterocolitis was 12.0 percent in the immune globulin group and 9.5 percent in the control group. CONCLUSIONS: Prophylactic use of intravenous immune globulin failed to reduce the incidence of hospital-acquired infections in very-low-birth-weight infants.

Artificial heart and assist devices: directions, needs, costs, societal and ethical issues.

A Working Group appointed by the Director of the National Heart, Lung, and Blood Institute (NHBLI) has reviewed the current status of mechanical circulatory support systems (MCSS), and has examined the potential need for such devices, their cost, and certain societal and ethical issues related to their use. The media have reported the limited clinical investigative use of pneumatically energized total artificial hearts (which actually replace the patient's heart) and left ventricular assist devices (which support or replace the function of the left ventricle by pumping blood from the left heart to the aorta with the patient's heart in place). However, electrically energized systems, which will allow full implantation, permit relatively normal everyday activity, and involve battery exchange or recharge two or three times a day, are currently approaching long-term validation in animals prior to clinical testing. Such long-term left ventricular assist devices have been the primary goal of the NHLBI targeted artificial heart program. Although the ventricular assist device is regarded as an important step in the sequence of MCSS development, the Working Group believes that a fully implantable, long-term, total artificial heart will be a clinical necessity and recommends that the mission of the targeted program include the development of such systems. Past estimates of the potential usage of artificial hearts have been reviewed in the context of advances in medical care and in the prevention of cardiovascular disease. In addition, a retrospective analysis of needs was carried out within a defined population. The resulting projection of 17,000-35,000 cases annually, in patients below age 70, falls within the general range of earlier estimates, but is highly sensitive to many variables. In the absence of an actual base of data and experience with MCSS, projection of costs and prognoses was carried out using explicit sets of assumptions. The total cost of a left ventricular assist device, its implantation and maintenance for a projected average of 4 1/2 years of survival might be approximately $150,000 (in 1983 dollars). The gross annual cost to society could fall in the range of $2.5-$5 billion. Ethical issues associated with use of the artificial heart are not unique. For individual patients these relate primarily to risk-benefit, informed consent, patient selection, and privacy. However, for society as a whole, the larger concern relates to the distribution of national resources.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin responses during catch-up growth of infants who were small for gestational age.

Growth characteristics of 15 full-term infants, selected because of weights more than 2 SD below the mean for gestational age, are described. The response to an intravenous injection of glucose was utilized to measure the insulin response of the infants at 6 months. Infants small for gestational age grow at a faster rate than appropriate-for-age infants during the first six months of life. There was a positive correlation between the growth velocity of the period and insulin release and a negative correlation between growth velocity and birth length. There was no correlation between these variables and increases in weight during the same period. Growth velocity during catch-up growth is related to the degree of preceding retardation but insulin may play a permissive role.