Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å

X-ray diffraction analysis of a crystal of HscA from Escherichia coli

A truncated form of HscA (52 kDa) containing both nucleotide- and substrate-binding domains has been crystallized and analyzed by X-ray diffraction. The crystal belongs to space group P212121 and diffracts to 2.9 Å

Structural Analysis Of Adp-Glucose Pyrophosphorylase From The Bacterium Agrobacterium Tumefaciens

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 Å, b = 93.79 Å, and c = 140.29 Å (α = β= γ = 90°) and space group I222. The A. tumefaciens ADPGlc PPase model was refined to 2.1 Å with an Rfactor = 22% and Rfree = 26.6%. The model consists of two domains: an N-terminal αβα sandwich and a C-terminal parallel β-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate. © 2008 American Chemical Society

Application of Nuclear Magnetic Resonance and Hybrid Methods to Structure Determination of Complex Systems

The current main challenge of Structural Biology is to undertake the structure determination of increasingly complex systems in the attempt to better understand their biological function. As systems become more challenging, however, there is an increasing demand for the parallel use of more than one independent technique to allow pushing the frontiers of structure determination and, at the same time, obtaining independent structural validation. The combination of different Structural Biology methods has been named hybrid approaches. The aim of this review is to critically discuss the most recent examples and new developments that have allowed structure determination or experimentally-based modelling of various molecular complexes selecting them among those that combine the use of nuclear magnetic resonance and small angle scattering techniques. We provide a selective but focused account of some of the most exciting recent approaches and discuss their possible further developments